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Part II, continued:

  The problem of microbial distribution in the soil is scarcely dealt with in the literature. We know almost nothing about the localization of microorganism in the soil. It is generally assumed that microbial cells are uniformly distributed in the soil penetrating all pores by diffusion. Therefore, upon quantitative calculation of seperate groups and bacterial species one usually limits himself to one or two soil samples.

  Such an assumption does not conform to reality, and the data so obtained lead to erroneous conclusions as to the distribution of the individual microbial species in the soil,

  Our studies show that the distribution of microbial cells in soil is not diffuse but focal. In each focus large or small cells of one species or several nonantagonistic species, grow and concentrate. Microbes, especially bacteria and mycobacteria inhabit soils in colonies (Krasil'nikov, 1936).

  The focal character of microbial distribution in the soil is being confirmed by the daily practice of microbiological soil studies. It to known that in one and the same field, Azotobacter, for example, may be found in one sample and not in another. If one takes 100-200 samples from 1 hectare of a given soil, cells of Azotobacter will not be found in all samples taken, depending upon the numbers of Azotobacter in this soil. The latter is determined by the state of the soil. In fertile, well-cultivated soils, rich in humus, Azotobacter will be found in every sample. In poor, nonfertile or virgin soils Azotobacter is rarely encountered in all the samples.

  Azotobacter was detected by us In all 200 samples taken from 1 hectare of a cultivated serozem soil under 3-year lucerne in Central Asia. In virgin soils we have found this microbe only in 3 samples (out of 2 00) and in poorly-cultivated soils in 45-85 samples. Similar results were obtained when studying the soils of the Volga area (chestnut soil and others) and the podsol soils. The soils of the Moscow Oblast' which were under forest until 5-10 years previously and now under various plants did not contain Azotobacter. We did not detect Azotobacter cells in any of the 1,250 samples studied. In garden soil this microbe was found in almost all samples (400 samples studied). In field soils (well -cultivated) Azotobacter was found in 60% of samples (860 samples were studied).

  For a more accurate determination of the focal distribution of Azotobacter in the soil we have carried out the following experiment (in the experimental stations near Moscow). One-hectare plots in three fields containing different numbers of Azotobacter were studied. On these hectare plots, 3 one-meter sectors, along the diagonal, were thoroughly analyzed for the presence of Azotobacter. To this end these sectors were divided into 100 small squares and 15-20 grams of soil were taken from each square. A total of 300 samples were analyzed from each hectare plot. The results of these analyses are given in Table 26, and the plan of Azotobacter distribution in the one-square-meter sectors is shown in Figure 54.

 

Figure 54. Schematic representation of the character of distribution of Azotobacter in soil. The sign "+" shows the presence of Azotobacter in 1 square meter soil sectors

a) poorly cultivated soil; b) well-cultivated soil (podsol, Moscow Oblast'). 1-10-numeration of squares.

  These data show that even in garden soil well-cultivated and systematically fertilized with mineral and organic fertilizers, Azotobacter cannot be detected in every sample tested. Of 100 samples taken from the 1-square-meter sectors, it was found in 90-96 samples. In field soils, well-cultivated and fertilized the number of samples containing Azotobacter was 34-60 and in poorly-cultivated soils, as well as in soils only recently under cultivation (5-10 years of cultivation, previously under forest) it was found in 8-16 samples out of 100.

  More detailed examination of the soil sample reveals small foci in which the Azotobacter cells are located. It is known from laboratory practice that when the soil sample is placed in small lumps on Ashby agar or gel plates, not each lump gives colonies of Azotobacter. The percentage of growth from each lump varies from 0%-100, depending on the soil (Figure 55). The method of placing soil lumps for the detection of Azotobacter and other species of bacteria (nitrifiers, cellulose decomposers, etc) is extensively used in microbiological practice.

Figure 55. Microfocal distribution of Azotobacter. Its content in soil lumps of 1 mg weight

a) soils rich in Azotobacter--all lumps contained Azotobacter cells; b) soils with moderate growth of Azotobacter, the number of lumps containing Azotobacter is large (on the average 40-60%); c) soils poor in Azotobacter, few lumps contain Azotobacter cells.

  We have analyzed samples of the well-cultivated soil of the same field as was the object of our previous experiments. Samples were taken from square-meter sectors, in the form of monoliths.

  Each sample after thorough mixing was divided into 5 samples of 0.1 g weight. Each such sample was divided into 100 small lumps, which were placed on the surface of Ashby agar (in Petri dishes). Five samples were taken from each of the sectors studied. The results are given in Table 27.

  The data presented in Table 27, show that the distribution of Azotobacter even in small lumps of soil is focal. The number of microfoci containing Azotobacter in such a lump is determined by the total number of Azotobacter in such soil and by other factors. In the samples studied by us, the lumps of 0.1 g contained in some cases from 21-99 and in others from 0 to 3-5 microfoci where Azotobacter could be detected (Figure 55).

  It should be noted that these foci are so small that they are not destroyed during ordinary mechanical crushing of the soil samples. In our experiments, we have carefully mlxed the soil samples and in spite of this, uniform distribution of Azotobacter was not achieved. The microfoci were thereby not destroyed or only a small fraction of them was destroyed. The percentage of lumps containing Azotobacter was almost identical to that of samples not subjected to mechanical crushing. In crushed samples 50-60 microfoci were found and in the intact noncrushed 30-50. Only when the lumps were ground into dust were the foci destroyed.

  The focal distribution and Azotobacter cell concentration described is also characteristic of other bacteria. It is well-defined in nitrifiers, cellulose decomposers, root nodule-bacteria, mycobacteria and others. It is less well-defined in fungi, and actinomycetes as a resuit of their biological pecularities. Rippel-Baldes (1952) noted focal development of Aspergillus niger in the soil. In square-meter sectors he found this fungus only in 14 squares out of a total of 100.

  The focal concentration of microbial cells in soils can directly be seen under the microscope, employing the method of Rossi-Cholodny, To this end cover glasses are immersed in the soil and after some period their surface is overgrown with microbes, bacteria, actinomycetes, fungi, yeasts and others.

  We have found colonies consisting of several cells of a size not greater than 10 µ, Clusters of microorganisms can be encountered which occupy an area of a 100 µ cross section. More frequently, colonies of moderate size (20-70 µ in diameter) and consisting of several tens of cells are encountered (Figure 56).

Figure 56. Distribution of microorganisms in soil

a) large colonies of Azotobacter; b) small colonies; c) general view on the distribution of microbes in the preparation (according to Vinogradskii, 1952).

  In our experiments we have noted the formation of colonies of Azotobbacter, sporiferous and nonsporiferous bacteria. Frequently, formation of colonies of mycobacteria, proactinomycetes and actinomycetes could be observed (Figure 57). Actinomycetes thereby form conidia with well-developed spores. Not infrequently actinomycetal hyphae develop into rodlike and coccoid cells, in the same way as can be observed on artificial nutrient media.

Figure 57. Colonies of actinomycetes in the soil

Branches with sporeforming cells are seen (spiral and straight). Many threads disintegrate into rods and cocci as in proactinomycetes (microphotos from glass imprints according to the method of Cholodny).

  The frequency of colony formation on cover glasses depends upon the soil properties. Especially great numbers of colonies are formed in the rhizosphere of plants. Bacteria and mycobacteria grow around thin root tips and around root hairs either in confluent layers as was noted earlier, or in separate foci, in formless clusters or in colonies,

  The formation of colonies in the soil has also been described by Cholodny (1934), Kubiena (1932), Rossi (1936), Vinogradskii (1952) and others. The microphotos given by us show clearly enough the concentrations of bacteria, fungi and actinomycetes.

  It is self-evident that together with the colonies on the cover glass, individual cells can be seen. They result from the destruction of the integrity of the colonies. The picture of microbial distribution in the soil is clearly seen under the microscope in ultraviolet light, especially after staining with acridine- orange. The individual cells and colonies of bacteria, actinomycetes and fungi standout sharply, glowing with a green color. Only a few cells glow with a red color. We have studied different soils according to this method, and always obtained positive results.

  In those cases when pores contain air, colonies of fungi and actinomycetes form fruit-bearing hyphae.

  In large pores, under favorable conditions, microbes proliferate and are concentrated in larger numbers than in the small pores. Bacteria, fungi and actinomycetes can penetrate adjacent pores through capillaries.

  Some investigators assume that microbes do not penetrate the very small pores of the soil. It was shown above that there exist organisms of ultramicroscopic dimensions lying on the border or beyond the border of visibility in optical microscopes (0.05-0.1 µ). To these belong phages (bacteriophages, actinophages), filterable bacterial forms, then certain cellular elements the so called L-forms, special regenerative bodies, etc of the ordinary species of bacteria and actinomycetes, and finally, small cells of individual organisms.

  Investigations showed that not only ultramicroscopic organisms can penetrate through the pores and small capillaries but also many bacteria and actinomycetes of normal size, of a diameter of 0.3-0.7 µ and more.

  It is known that bacteria and actinomycetes do not pass through filter candles upon ordinary filtration under pressure. Sterilization by filters is based on this observation., In laboratory practice the most widely used filters are the Berkefield filter (L₃ L₅), Chamberlain filter (N); Seitz filters or membrane filters with pores of a definite size.

  If such filters are filled with liquid bacterial suspension and immersed in a nutrient solution (meat-peptone broth), then after an incubation period at 25-37° C, cells will pass through their walls and start growing outside them. In our experiments the following microorganisms passed through such filters: Bact. coli, Bact. prodigiosum,.Ps. aurantiaca , larger sporiferous species, Bac. mycoides , Bac. mesentericus, Bac. megatherium, actinomycetes. A.violaceus, A. coelicolor, and A. globisporus.

  Besides bacteria, actinomycetes and fungi in the course of growth pass comparatively easily through small-pore clays. We studied the clays of natural deposits underlying the soils of the Moscow district fields. Clay samples were placed in Koch dishes, wetted with water uniformly mixed and distributed in a layer of 1.5-2.0 cm. Bacteria were introduced into the center well. After incubation at 25° or 37° C samples, from different distances from the center well, were taken at various time intervals and analyzed. The experiments were carried out in sterile and nonsterile conditions. In nonsterile conditions easily detectable microbial species were employed: Bact. prodigiosum , Ps. aurantiaca, Bact. coli, Az. chroococcum, Bac. Mycoides, A. violaceus. In sterile experiments, besides the afore-mentioned Bac. mesentericus, Ps. fluorescens was also employed. The results of these experiments are given in Table 28.

  As can be seen from the data, the speed of growth and bacterial mobility is not the same in the different bacteria and actinomycetes and varies in relation to the properties of the clay. The threads of actinomycetal mycelium move with the greatest velocity. Some nonsporiferous bacteria grow rapidly. Cells of Bact. coli grow slowly and Bac. mycoides is the slowest of them all.

  The microbial cells do not move through small pores of the natural and artificial substrates mechanically, or under the action of external pressure, as they do under filtration, but they move as a result of overgrowth. Dead cells do not pass through filters. There is no movement of cells, or only very weakly, when filters with living bacteria are immersed in pure water.

  The more intense the growth of microbes, the more rapidly they pass through small pores and capillaries. The optimal temperature for the growth and multiplication of bacteria Is, at the same time, the most favorable for their passage through small pores. At a temperature of 5-7° C Bact. coli and Bact. prodigiosum multiply slowly and pass through a Chamberlain candle in 60-80 hours. At a temperature of 25° C this period is shortened to 20-30 hours.

  The motility of microbes in clay is increased upon introduction of organic substances into the clay as meat- peptone broth, or saccharose. To do this, a well is made in the clay which is prepared in the same way as in the preceding experiment, and, at some distance from it, an elongated groove is dug. Bacteria are placed in the central well (aqueous suspension), and nutrient substances (those listed above) in the groove. It was noted that microbial cultures of Bac. mycoides, Bact. prodigiosum, Ps. aurantiaca, and A. coelicolor moved in the direction of the nutrient broth quicker than in the control experiments. In one day Bact. prodigiosum moved, in the control experiment, 1.5-2.0 cm, in the presence of the meat-peptone broth 3.0-3.5 cm, in the presence of saccharose 2.5-3.0 cm. Bac. mycoides in the control moved 0.7 cm and in MPB 1.5 cm; Ps. aurantiaca moved 2.0 cm in the control as compared with 3.0-4.0 cm in the experiment. The movement of actinomycetes in the presence of organic substances was by 1.0-1.5 cm greater than in the control. It should also be pointed out that the degree of permeability of small porous substrates also depends on a great number of external factors, such as the pH of the medium, aeration, and the composition of the soil solution. Anything that favors the growth and proliferation of organisms also enhances the overgrowth of the substrate.

  Thus, the data obtained established the possibility of penetration of microbial cells through the smallest soil pores, under natural conditions.

  Apparently no soil pores exist which cannot be penetrated by microbes.

  In the chapters devoted to structure and growth of bacteria, it was shown that microorganisms may exist under laboratory conditions in a polymorphous state. Along with normal or ordinary cells there exist many forms of bacteria and actinomycetes which deviate from the norm in size and in form.

  In which form do these microbes exist in the soil? What is their cellular form, and is the polymorphism of cells in the soil as characteristic as in the artificial cultures?

  These problems are very little dealt with in the literature. If our knowledge on the quantitative and qualitative composition of soil microbes is slight, then the problem of the forms of the soil microbes is even less known.

  It should be recalled that during direct counting of soil smears according to Vinogradskii, or overgrown glass according to Cholodny, not infrequently, the usual forms of bacteria, actinomycetes, fungi, protozoa and others were seen.

  Consequently, the cells of microorganisms in the soil are of the same form and size as those grown on artificial media.

  But there are instances when the smears enriched in soil microbes, as well as the overgrown glass do not contain the usual microbial cells, or they contain them in small numbers only.

  We purposely enriched the soil with microbes before the analyses. To this end in one series of experiments we have introduced into the soil such nutrients as sugars and meat-peptone broth. Subsequent inoculation on agar media revealed hundreds of millions of bacterial cells in the analyzed sample. An enormous number of bacteria was found by using the method of serial dilutions (10-30 millions per gram of soil). With such a large number of bacteria in the smears on Vinogradskii's glass, we should have been able to detect them in the microscopic field (lens 90, ocular 10) in hundreds and thousands, while in reality their number did not exceed 30-50 and more often there were only 10-20 cells*. (* The determination of the number of the bacteria was carried out as follows: a 4 cm² square was drawn on a slide, One drop of soil suspension (0.05 ml) was uniformly spread an the square. The smear was air-dried and stained with fluorochrome dyes. It was studied under the microscope with lens 90, oc. 10. The number of fields was calculated by the following simple calculation. The radius of a microscopic field = 85 µ, its area--3.14 x 85 µ = 22,686.5 µ². Since there are 100,000,000 µ² in l cm, then in the given area there are 4,402 fields.)

  Two to three days after the introduction of MPB into the soil, a large number of cells in the form of rods (150-200 in a microscopic field) could be seen in smears and in the glasses of overgrowth. These cells are mostly very small and apparently belong to the group of nonsporiferous bacteria of the genus Bacterium and Pseudomonas. Not infrequently, cells of the mycobacterium type are encountered, larger cells of the sporiferous bacteria are very rarely found. As a rule, the latter are without spores.

  Three to five days after the introduction of the afore-mentioned broth, the bacteria seem to vanish and are no longer detectable in the smears or on the glasses of overgrowth. At the same time hundreds of millions of bacteria can be detected upon inoculation on nutrient media. Consequently, no less than 100 cells ought to have been seen in the microscopic field upon the examination of such slides.

  In the other series of our experiments pure cultures of Ps. fluorescens, Bact. prodigiosum and Az. chroococcum, grown on artificial nutrient media, were introduced into the soil. The amount of the bacteria introduced was 100-1,000 million cells per gram of soil. The analyses were made by the method of direct counting. Already after one or two days no cells of the first two genera could be detected. At the same time millions of them could be detected upon inoculation of nutrient media. Azotobacter was detectable for 3-6 days upon inoculation in artificial media; in smears the cells of Azotobacter were found in larger numbers, but they were all dead. They did not grow in nutrient agar and did not change their form for long periods of time, as If they were in a state of fixation. Their protoplasm became less dense and the disappearance of the reserve food was sometimes observed.

  The number of denitrifying bacteria in the rhizosphere of lucerne under the conditions of the Vakhsh valley reaches 10⁹-10¹¹ cells per gram, whereas we have found only individual cells in the smears.

  We have studied the rhizosphere of pea and corn, grown in special containers filled with soil or quartz sand. When the plants grew in the sand all the cellular elements of bacteria, fungi and actinomycetes growing around the plant roots were clearly seen in the imprints on the glass. Bacterial cells present near the roots and also on the roots and on the root hairs were of the same form and size as those seen after growth in artificial media. Cells of nonsporiferous and sporiferous bacteria, mycobacteria, proactinomycetes and actinomycetes were clearly seen (Figure 58). The imprints. clearly revealed cells of Bac. megatherium, Azotobacter, and other bacteria of characteristic cell structure.

Figure 58. The growth of microorganisms in sand, in the root zone of corn:

a) Azotobacter, introduced from the outside; the cells are dispersed around a small sector of the root; b) Azotobacter, small cells growing in colonies; c) nonsporiferous bacteria; d) sporiferous bacteria; e) colonies of fungi with fruiting branches.

  While analyzing the imprints of the same plants, but grown in soil, we almost failed to detect normal bacterial cells. Individual clusters, or small colonies of cells of coccoid form were infrequently observed. Frequently threads of actinomycetes and fungi were seen. Inoculation of this rhizosphere soil by the method of sterile dilution revealed hundreds of millions of cells. Analogous analysis of soil in smears should have revealed not less than 200-300 cells per field; however, almost no cells were seen on using this method.

  The disappearance of the bacterial cells that were introduced into the soil was observed by Dianova and Voroshilova (1925). Chudiakov (1926) explained this phenomenon by the adsorption of bacterial cells by soil particles. No doubt, there is adsorption of bacterial cells in the soil but not in such proportions, furthermore this process is of quite a different character.

  Novogrudskii et al. (1936) gave much attention to the problem of the state of bacterial cells in the soil. They introduced slides containing bacterial cells into the soil and studied them under the microscope after various periods of time. In this way they found that the bacterial cells undergo deformation, degeneration, autolyzation and disappear.

  Vinogradskii (1952) and many other investigators noted the presence of a large number of coccoid cells in the slides of soil smears, taking them for micrococci. According to our observations, the number of cocci in the soil is small and the coccoid cells are coccoid forms of other microbes.

  By employing different methods of investigation, including the method of overgrowth, we were able to establish that these cocci are most frequently the cells of mycobacteria, proactinomycetes, and mycococci; nonsporiferous and sporiferous bacteria may also often exist in such a form.

  Minute corpuscles in the form of debris can always be detected in smaller or larger numbers during the examination of soil smears. These corpuscles strongly adsorb dyes. We assume that this granular mass consists mainly of soil colloids and partly of decomposition products of microorganisms. It has been proven experimentally that they contain cell gonidia. So, if the smear or the imprint on the glass as well as the glasses of overgrowth, are covered with a thin layer of nutrient agar (or better with Chapek agar or Ashby agar) and placed in a moist chamber at 18-20° C, then, after some time, minute colonies of germinated gonidia can be detected.

  'The formation of numerous colonies was observed in the absence of well-defined cellular forms in the smears. At the same time the majority of forms, thought by us to be cells, did not grow.

  Our investigations as well as the investigations of Novogrudskii, show that the ,cells introduced Into the soil are subject to various deformations. The majority of the cells degenerate. In so doing they swell slightly, their protoplasm becomes granulated, lightens and after the dissolution of the membrane, disappears altogether. Small granules and cell debris remain. Other cells begin to divide but do not elongate and the daughter cells in their turn divide without corresponding growth. As a result, minute cell elements are formed in the form of small granules or corpuscles.

  Such cell division was observed in the sporiferous bacteria, Bac. megatherium, Bac. mesentericus and Bac. subtilis, in mycobacteria, in certain strains of nonsporiferous bacteria of the genus Pseudomonas and in Bacterium, in root-nodule bacteria (Rhizob. trifolii) and in Azotobacter. In the latter, the decrease in size of the cell due to cell division without concomitant cell growth is quite often observed (Figure 59). Great changes in the cellular elements are observed in the nonsporiferous bacteria (Figure 60).

Figure 59. The development and transformation of the bacteria cells in soil (podsolic) in the rootzone. Azotobacter introduced from the outside

a) few cells remained unchanged (dead cells); b) most of the cells are of much diminished sizes (dimensions) with weakly refracting plasma; c) on some slides the Azotobacter cells are of small sizes with dense plasma; d) a colony of transformed Azotobacter cells (marked by x).

 

Figure 60. Transfor mation of bacterial cells in the soil into minute forms. The cells transform into the state of small granular elements--conidia--and proliferate in this state

a) Bac. megatherium; b) Bac. mesentericus; c) Pseudomonas sp., normal cells on a medium containing soil extract; d) cells introduced into the soil, small and deformed; small granular conidia of Pseudomonas sp., are inside the circle; among them are large oval-coccoid cells of other microbes.

  These small cells preserve their viability to a certain degree. In favorable conditions, on artificial media, they grow, giving normal offspring.

  We observed transformations of bacterial cells during the decomposition of plant residues (roots or parts growing on the surface). At first the bacteria grow together with other microbes in the usual rodlike forms. After a certain period of time, when the vegetative residues have to a certain extent decomposed, the bacteria degenerate and disintegrate with the formation of a mass composed of small granules. This mass contains many gonidial forms and small cellular elements.

  In such a manner it can be shown that, under natural conditions, bacteria have different forms of existence. In addition to those forms, which, from our point of view, are the ordinary forms, they also most frequently exist in the form of minute cellular elements, as coccoid cells of decreased size or In the form of small gonidial corpuscles. These forms lead an independent existence for an unlimited period of time. Only under special conditions (excessive nutrition, etc) do they assume a rodlike form and a size characteristic of each species.

  Many if not all actinomycetes also exist in the soil in cellular forms differing from those in the artificial nutrient media. In nutrient medium they form a well-developed nonfragrmented mycelium, whereas in the soil their mycelium is frequently fragmented. Transverse partitions are formed in its threads, with a resultIng fragmentation into rodlike elements (Figure 54).

  On the glass of overgrowth one can observe the successive changes in one and the same colony of actinomycetes and the formation of threadlike, rodlike and coccoid cells. This process is similar to that observed in proactinomycetes. (Krasil'nikov, 1938a). Hyphae or their branches frequently become fragmented with subsequent rounding and the formation of oval or spherical cells.

  Actinomycetes in the soil grow either as actinomycetes, proactinomycetes or even as mycobacteria. Their colonies resemble the colonies of proactinomycetes or mycobacteria. However, by the use of the method of covering the glass of overgrowth with a layer of nutrient agar, such colonies grew and gave the characteristic culture of actinomycetes (Act. globisporus).

  The process of fragmentation of threads into rodlike and coccoid cells can also be induced in the actinomycetes on nutrient media, if they are grown with constant shaking (on shakers) or during submerged growth (in fermenters).

  Analogous transformations were not detected by us in fungi. Apparently, they preserve their mycelial structure in the soil. However, the possibility of sharp changes and transformations of individual species of this group of microorganisms cannot be precluded.

  When considering the problem of the distribution of microorganisms in soils, the climatic or geographical conditions, in other words the ecological and geographical factors, should be taken into account, It is well known that there is no place on earth where microorganisms could not be found. They can be found in the extreme points of the Arctic and Antarctic. In places where the earth thaws even for short periods, it is populated to a greater or lesser extent with microbes. They exist in dry, hot steppes and deserts, in naked sands and rocky terrain, in valleys and on mountain summits.

  Microorganisms also grow on the surface of eternal snow, often covering it with a thick layer of bright colors.

  The soil-climatic conditions of existence cannot but reflect on the qualitative composition of the microflora. Microbial species change their natural properties in the process of their adaptation to the external conditions.

  The available data on the regularity of distribution and formation of microbial coenoses in the soil are scarce and they deal with only a few genera : Azotobacter, root-nodule bacteria, Bac. mycoides, and some other well-described bacteria.

  A great number of works are devoted to the distribution of Azotobacter in the soil. Attempts were made to determine some regularities of the ecological order involving this genus (Sushkina, 1949).

  This problem was given attention in the works of Mishustin (1947). From a study on the distribution of the sporiferous bacillus Bac. mycoides in soils of the USSR, the author gives numerous data collected by him during many years. According to his data, the microorganisms are distributed in the same way as the higher plants in strict relation with the geographical location. Certain species exist in the North and others in the South. According to the author, microbes change their biological properties according to the geographical conditions.

  Mishustin's scheme of the zonal distribution of microbial species in soils, is only a first attempt to establish regularity in the distribution of microorganisms and therefore it is of great interest. It needs, however, essential corrections and a number of statements require verification by factual data.

  Before one can speak of regularity of microbial distribution, one must possess accurate data on distribution of individual species, or in other words a chart of microbial distribution. For the compilation of such a chart numerous analyses of soils of every region and every geographical zone are necessary. Such analyses are, not at present available.

  This problem becomes complicated by the fact that the analysis can be made only in regard of a few well-known species. The majority of microbes, especially among the bacteria cannot be distinguished from each other. The inability to distinguish accurately, by morphology, one species from another, narrows the scope of microbiological studies of an ecological and geographical character. One has to limit oneself to a few species.

  Another difficulty in the compilation of a geographical chart of microorganisms lies in the fact that individual bacterial species are distributed in the soil, not diffusely, but in separate colonies and foci and, in addition, they often grow and can be detected only in certain seasons.

  The number and the size of the foci or individual concentrations of microbes also depend on the kind and state of the soil, as well as upon the season and other conditions. Sometimes, analyses have to be carried out in many dozens or even hundreds of samples, in order to be able to detect the presence of this or another microbe, and the degree of its growth. This explains the variability of the data on the distribution of Azotobacter or other bacterial species in one and the same soil.

  It should be noted that it is much more difficult to establish the habitation areas of microorganisms than that of plants.

  It is well known that the distribution of plants is characterized by more or less sharply pronounced localization of species. The study-of plant localization is the basis of plant geography (Alekhin, 1950).

  Among higher plants there are species widely distributed over the earth. These are the cosmopolites. Other species are present in restricted areas. These are the "stenochore" species. Among them there are plants which can be found only in a few localities. They are called the endemics.

  When considering the microorganisms we cannot say whether there are among them endemics and stenochores--adapted for growth in certain restricted geographical zones. Microbes are known which live in hot springs, the temperature of which exceeds 60-70° C. Microbes maybe encountered in a milieu saturated with H₂S, CH₄, and other substances. These microorganisms are endemics, restricted to the ecological zone. No data are available in microbiology on the existence of a strict differentiation of the microflora of tropic, subtropic and arctic soils. The well-known species of microbes can be detected in all the geographical zones, tropical and polar, For example, the Azotobacter, Az. chroococcum thrives in soils of the extreme North (Igarka, Yakutiya, Arkhangel'sk Oblast', Kola Peninsula, Severnaya Zemlya, etc), In the tropics and subtropics (Trans Caucasus, India, Australia, Egypt, etc). Bac. mycoides, Bac. megatherium and Bac. mesentericus can be encountered in all geographical zones. The root-nodule bacteria of astragals and clover are found in the soils of Central Asia, Caucasus, Crimea, the moderate belt of the RSFSR and also in the soil of the Kola Peninsula, the islands of the Arctic Ocean, Severnaya Zemlya, Franz Joseph Land and others. (Kriss, Korenyako and Migulina, 1941; Kazanskii, 1930). The root-nodule bacteria of southern plants--soya, Onobrychis and lucerne can be found in the soil of the Leningrad Oblast', Kola Peninsula and Moscow Oblast',

  For a long time the yeast Schizosaccharomyces octosporus was considered in the mycological literature to represent the typical population of the southern microflora. We have, however, found them In many localities around Leningrad, in the sap of the oak.

  Of the actinomycetes, such well -known species as A. globisporus and A. streptomycini are found in the soil of the Kola Peninsula, in the Moscow Oblast' and in the Caucasus, in the Crimea and in Central Asia. The violet actinomycete A. violaceus and the blue A. coelicolor live in soils of the Vakhsh valley, Crimea, Caucasus, Moscow Oblast', Igarka, Astrakhan' and Leningrad. According to data in the literature, the same species live in soils of Latin America, Australia, Japan, Italy, and other southern countries.

  All these data support the hypothesis that the known microbial species are cosmopolites.

  Certain representatives of physiological groups of microorganisms are even more widely distributed. Thermophiles and psychrophiles can be detected in considerable numbers everywhere, In the Far North, in the tropics, on mountain summits and in valleys (Egorova, 1938, Mishustin, 1945b, Kosmachev, 1955).

  Hydrophiles, xerophiles, nitrifters, denitrifiers, cellulose decomposers, root nodule bacteria and others live in soi1. An impression is created that all these organisms are cosmopolites. However, a more detailed study shows this to be wrong. The afore-mentioned organisms do not represent individual species but heterogenous groups. In each group there are species sharply differing from each other. Some of them are widely distributed, others restricted to certain localities.

  In medical microbiology pathogenic bacteria are known, whose distribution is restricted to some countries. Among the saprophytes there are forms, some of which are adapted to a warmer climate, others to a lose warm or even to a cold climate.

  Is their distribution caused by geographical or ecological factors?

  In our opinion, the geographical locality by itself is not a factor. Every area or space populated by microbes is characterized by many external conditions. Temperature, light, humidity, aeration, winds, soil composition, etc. All these conditions are, to a certain degree, determined by the geographical locality; only through these conditions, may the locality affect the biology of the living organisms. Consequently,, the regularity of microbial distribution is determined by ecological factors and not by geographical locality as such.

  Different external factors will predominate in each place of bacterial habitation. When the localities are considered from the point of view of latitudes, then the dominant factor will be, in all probability, the temperature. The same factor will also predominate with the altitude.

  Assuming the temperature to be of the utmost importance, many authors attempted to establish the regularity of microbial adaptation in nature according to this factor. Mishustin brings forward material on the correlation of microbial distribution to the temperature of the zones in which they are found. According to his data, Bac. mycoides of northern soils grows at a lower temperature than the strains of the same species isolated from southern soils. The optimal temperatures for growth become lower with the latitude. For example, the optimal temperature for growth of bacteria in the Crimean soils is 36- 38° C. The maximal temperature at which their growth is I arrested Is 44-45° C. The optimal temperature for the growth of bacteria in the Batumi soils is 38° C and the maximum is 46° C; In the soils of

Kursk Oblast'; Optimal ° C 33-35, Maximum ° C 43-45
Moscow; Optimal ° C 29-31, Maximum ° C 42
Leningrad; Optimal ° C 29-31, Maximum ° C 40
Arkhangel'sk; Optimal ° C 27-30, Maximum ° C 36-39

  The temperature as an ecological factor effects the total number of psychrophiles and thermophiles. Investigations show, that thermophiles are encountered in arctic and subarctic soils but in a lesser amount than in the southern soils. For example, in the islands of the Arctic Ocean, Severnaya Zemlya and Franz Joseph Land, thermophiles growing at 55° C contain about 1-2 cells per 100,000 mesophiles; in the soils of Crimea (southern shore under vine) and in the soils of the Caucasus (krasnozems under tea plantations in Anaseuli) the number of thermophiles increases 10-50 times.

  It should be noted that the bulk of the microflora of northern and southern soils consists of mesophiles, growing within the temperature range of 3-5 to 35° C. We have carried out special experiments on the comparison of the microflora of soils of the northern, southern and moderate belts. The soils were inoculated into MPA, Chapek and Ashby agar media. The inoculation was at 3-4, 10, 20, 25, 30, 37 and 42° C. The results are given in Table 29.

  Most bacteria have a different minimal temperature of growth, depending on the place of isolation. Cultures isolated from the soil of Franz Joseph Land have a minimum at 3° C and their growth starts after 10-12 days; those from the soil of Moscow district start to grow after 15-17 days and bacteria from the Crimea and Caucasus start to grow after 18-20 days at the same temperature. The maximum temperature of bacterial growth is even more sharply pronounced. Most bacteria from the soils of Franz Joseph Land and Severnaya Zemlya cease to grow at 30-32° C. At a higher temperature only individual organisms grow, from 100 to 1,000 cells per gram of soil. Among the bacteria from Moscow soils there are about 100,000 cells per gram of soil which can grow at 32° C. There are many forms that can grow at 42° C. In the southern soils of Crimea and Caucasus the number of thermophiles is even higher.

  The given data show that in all geographical localities the mesophiles prevail. They are the forms which are of the greatest importance in the processes taking part in the soil.

  The behavior of the soil microflora toward higher temperature is even more sharply differentiated. The further south is the soil, the more thermophIles and actinomycetes can be found. Apparently this part of the microflora can serve as an index in geographical distribution and formation of biocoenoses. However, this problem needs to be further studied.

  The essential factor in the ecology of microorganisms is the humidity of the climate and the soil. The characteristic and general feature of marshy soils of any geographical zone is their supersaturation with moisture. The latter creates conditions of anaerobiosis, which lead to the formation of the corresponding microbial biocoenoses. In such soils anaerobes are the characteristic and prevailing form.

  In dry soils of the southern deserts and steppes the lack of moisture creates special conditions for the growth and formation of xerophytes. These forms are frequently found among mycobacteria and actinomycetes. According to our observations in the Volga steppes, in the Kara Kum desert and in other. dry regions, mycobacteria prevail in periods of low humidity.

  Saline soils are inhabited by halophytes, organisms which can tolerate salt, including many groups of bacteria, mycobacteria and actinomycetes. Sushkina (1949) described a halophyte form of Azotobacter. The halophils can be encountered among the sporiferous and nonsporiferous bacteria. They can be isolated from normal nonsaline soils, but their number in the latter is smaller than that in saline soils. In normal soils only single cells can be detected, whereas in the saline soils they reach tens and hundreds per gram.

  Under other soil and climatic conditions other factors exist which influence the biology and composition of the microbial population.

  It is self-evident that in each case not one single factor is operative but an intricate complex of factors, determining the formation of the microbial population. The predominant factor is accompanied by others less important, but characteristic for each locality.

  One of the main reasons for our ignorance of the ecological geographical distribution of microorganisms is the difficulty in the determination of the individual species. This is made more difficult by the variation and polymorphism of the bacteria, 'The degree of polymorphism, even of the better known bacteria, is unknown. For example, Bac. mycoides, Bac. mesentericus and others may give 7-10 different variations differing from each other to a greater or lesser extent.

  As noted above, one and the same culture of Bac. subtilis, Bac. mesentericus, Bac. cereus or other microorganisms can be detected in the soil in various forms.

  The colonies of these bacteria are either of the typical mesenteroid form or undulate-smoothly or undulate-granularly with edges, even, wavy, or otherwise. Some of them resemble Bac. cereus, others Bac. brevis, still others Bac. vulgaris, etc.

  A granular form of Bac. mycoides is not infrequently encountered. In many soils (podsols, serozems and others) together with typical mucoid and actinomycetal forms (Crimea soils). In the chestnut soils of the Trans-Volga region only the granular variant in encountered (Krasil'nikov, Rybalkina, Gabrielyan and Kondrat'eva, 1934).

  Not knowing the origin of the variants, one may think them to be independent species, as indeed frequently happens.

  A reverse phenomenon takes place in some other species. Different variants may have the same external appearance and the differences in their physiological and biochemical properties are not easy to determine. It is very difficult to distinguish such variants. Such forms are encountered in Az. chroococcum and in many species of the genera Bacterium and Pseudomonas.

  It was shown in the chapter on variations that the species Az. chroococcum is a group of organisms consisting of sufficiently diverse cultures representing separate taxonomic entities, strains, forms, varieties or even species ("sufficient" to justify further taxonomic division).

  The genera Bacterium, Pseudomonas and others are even more diverse. The inadequacy of the methods of differentiation of these organisms does not enable us to detect this diversity. Bacteria taxonomically belonging to a single species in reality represent whole groups, which in turn should be subdivided into separate taxonomic entities.

  Differentiation of such species or groups in actinomycetes can be accomplished. By employing the method based on the specificity of antagonism, we were able to disclose the complexity of some species of monolithic taxonomic entities which were designated as species. For example, the former species A. globisporus is now divided into approximately 10 species and A. coeliccolor into 7-8 sufficiently easily differentiated species, etc.

  All this supports the hypothesis that there is much greater diversity in nature than can be revealed by our methods. This diversity is caused by species variability and adaptation to different environmental conditions.

  In what manner are all these forms distributed in the soil? Are they distributed according to geographical zones, or according to ecological conditions? Investigations show that the different forms, variants and even species can be frequently detected in the same soil and in the same sector. For example, root-nodule bacteria, of clover in the podsol soil near Moscow have many diverse forms and variants which differ from one another by their cultural, physiological and biological properties. Among the 150 cultures isolated from the soils of the experimental fields of the Academy of Agriculture im. Timiryazev and from the soil of the Experimental Station of the Moscow State University in Chashnikovo, more than 20 variants were detected.

  Such variants were observed among the root-nodule bacteria of lucerne, isolate from the serozem soil of the Vakhsh valley (Tadzhik SSR) Two hundred strains of root-nodule bacteria of lucerne were isolated from the soil of two regions of Azerbaijan and studied. Among them more than 50 variants were found. These variants differed sharply from one another. Strains which differed from each other even more, were detected among the root-nodule bacteria of Onobrychis isolated from the very same sector. Different forms and variants of Az. chroococcum can be found in one field. In Moldavian soils 10 forms each differing from the others were found (Babak 1956). No loss a diversity of strains may be seen in the soil of Latvia (Pavlovich, 1053), In the Kola Peninsula (Ezrukh, 1956), in (Soviet) Central Asia In the Crimea and In the Caucasus (author's observations). Petrenko (1953) detected a large number of different forms of Azotobacter, in podsolic soils near Moscow.

  What is the cause of such diversity? In our opinion it is the action of microecological factors or microzonality in the soil. No soil is uniform in its properties. It consists of microfoci or foci which differ from each other in the content of nutrients, humidity, temperature, composition of the microflora, vegetation, etc. The influence of the microorganisms and especially of antagonists, on the growth and development of now forms and individual species, is very great. Man's activity and the vegetative coverage are of great importance in the formation of microbial biocoenosis.

  The cultivation of the soil changes its microbial composition. The draining of marshes favors the growth and concentration of aerophiles and xerophiles. Irrigation of waterless deserts increases the number of hydrophiles. The cultivation of soils of northern regions or mountain summits transforms the microflora of those soils.

  It is known that many virgin soils do not contain Azotobacter; but it is sufficient to plow and cultivate them in order to enable this microorganism to grow. In chestnut soils of the Trans-Volga region Azotobacter cannot be detected, but it appears there immediately after the soils have been irrigated. Fertilization of soils, especially with organic fertilizers, sharply increases the growth of this organism in the podsol soils.

  As already pointed out, the better the soil is cultivated, the more microorganisms it contains and the higher its fertility. With cultivation, the number of microorganisms increases. The increase in number involves such genera as Pseudomonas , Bacterium, Azotobacter, sporiferous bacteria, actinomycetes, fungi and others. The thermophiles, mesophiles, aerobes, anaerobes, antagonists, activators, and many others also increase in number. It is difficult to say which of those microorganisms is the best indicator of a cultivated soil.

  Moshustin (1945a, 1947) suggested that the thermophilic bacteria be used as an indicator of the degree to which the soil in being cultivated. The author* thinks that these organisms are introduced into the soil together with organic fertilizers. * [This is ambiguous in the Russian text but probably refers to Mishustin.]

  According to our investigations the thermophilic organisms, bacteria as well as actinomycetes, are natural inhabitants of the soil. Their numbers increase with the degree of cultivation of the soil. The other organisms proliferate at the same time. In some instances the thermophiles increase under more intense cultivation of the soil and sometimes other groups of bacteria and actinomycetes predominate.

  Thermophiles can serve as an indicator of soil cultivation to the same extent as many other organisms or groups.

  To our mind Azotobacter is the best indicator of the intensity of soil cultivation.

  Thus, in order to draw up a chart of the distribution of microorganisms in soils, detailed information on the growth of individual species in each region, or even in each field during different seasons of the year is needed. Such data are not available at present and those which are, cover restricted areas only. An immense and painstaking work is called for, a work which can only be performed by a large team of microbiologists, working in different institutions but according to one plan and using the same methods.

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