HOME      AG LIBRARY      KRASILNIKOV TABLE OF CONTENTS


Part II, continued:

 

Distribution of Microorganisms in Soil

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

  Such an assumption does not conform to reality, and the data so obtainedlead to erroneous conclusions as to the distribution of the individual microbialspecies in the soil,

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

  The focal character of microbial distribution in the soil is beingconfirmed by the daily practice of microbiological soil studies. It to known thatin one and the same field, Azotobacter, for example, may be found in one sampleand 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 uponthe numbers of Azotobacter in this soil. The latter is determined by the stateof the soil. In fertile, well-cultivated soils, rich in humus, Azotobacterwill be found in every sample. In poor, nonfertile or virgin soils Azotobacteris rarely encountered in all the samples.

  Azotobacter was detected by us In all 200 samples taken from1 hectare of a cultivated serozem soil under 3-year lucerne in Central Asia. In virginsoils we have found this microbe only in 3 samples (out of 2 00) and in poorly-cultivatedsoils in 45-85 samples. Similar results were obtained when studying the soils ofthe Volga area (chestnut soil and others) and the podsol soils. The soils of theMoscow Oblast' which were under forest until 5-10 years previously and now undervarious plants did not contain Azotobacter. We did not detect Azotobactercells in any of the 1,250 samples studied. In garden soil this microbe was foundin almost all samples (400 samples studied). In field soils (well -cultivated) Azotobacterwas found in 60% of samples (860 samples were studied).

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

Table 26
Distribution of Azotobacter in the soil
(number of samples containing Azotobacter in %)

Sectors in fields

Sector I

Sector II

Sector III

Average

Garden soil

92

90

96

92.7

Field, well-cultivated, under 3-year clover

53

60

34

49

Field, poorly cultivated, under 3-year clover

16

8

12

12

  These data show that even in garden soil well-cultivated and systematicallyfertilized with mineral and organic fertilizers, Azotobacter cannot be detectedin every sample tested. Of 100 samples taken from the 1-square-meter sectors, itwas found in 90-96 samples. In field soils, well-cultivated and fertilized the numberof 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, previouslyunder forest) it was found in 8-16 samples out of 100.

  More detailed examination of the soil sample reveals small foci inwhich the Azotobacter cells are located. It is known from laboratory practicethat when the soil sample is placed in small lumps on Ashby agar or gel plates, noteach lump gives colonies of Azotobacter. The percentage of growth from eachlump varies from 0%-100, depending on the soil (Figure 55). The method of placingsoil 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 fieldas was the object of our previous experiments. Samples were taken from square-metersectors, in the form of monoliths.

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

Table 27
Azotobacter distribution in soil samples
(growth from lumps, %)

No .of sample

Sector I

Sector II

Sector III

1

23

70

28

2

99

39

15

3

97

55

31

4

88

86

8

5

56

42

21

Average

72.6

58.4

20.6

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

  It should be noted that these foci are so small that they are notdestroyed during ordinary mechanical crushing of the soil samples. In our experiments,we have carefully mlxed the soil samples and in spite of this, uniform distributionof Azotobacter was not achieved. The microfoci were thereby not destroyedor only a small fraction of them was destroyed. The percentage of lumps containingAzotobacter was almost identical to that of samples not subjected to mechanicalcrushing. In crushed samples 50-60 microfoci were found and in the intact noncrushed30-50. Only when the lumps were ground into dust were the foci destroyed.

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

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

  We have found colonies consisting of several cells of a size not greaterthan 10 µ, Clusters of microorganisms can be encountered which occupy an areaof a 100 µ cross section. More frequently, colonies of moderate size (20-70µ in diameter) and consisting of several tens of cells are encountered (Figure56).

 

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 therebyform conidia with well-developed spores. Not infrequently actinomycetal hyphae developinto rodlike and coccoid cells, in the same way as can be observed on artificialnutrient 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 thesoil properties. Especially great numbers of colonies are formed in the rhizosphereof plants. Bacteria and mycobacteria grow around thin root tips and around root hairseither in confluent layers as was noted earlier, or in separate foci, in formlessclusters 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 microphotosgiven 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 ofthe colonies. The picture of microbial distribution in the soil is clearly seen underthe 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 studieddifferent soils according to this method, and always obtained positive results.

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

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

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

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

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

  If such filters are filled with liquid bacterial suspension and immersedin 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 experimentsthe 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 growthpass comparatively easily through small-pore clays. We studied the clays of naturaldeposits underlying the soils of the Moscow district fields. Clay samples were placedin Koch dishes, wetted with water uniformly mixed and distributed in a layer of 1.5-2.0cm. Bacteria were introduced into the center well. After incubation at 25° or37° C samples, from different distances from the center well, were taken atvarious time intervals and analyzed. The experiments were carried out in sterileand nonsterile conditions. In nonsterile conditions easily detectable microbial specieswere employed: Bact. prodigiosum , Ps. aurantiaca, Bact. coli, Az. chroococcum,Bac. Mycoides, A. violaceus. In sterile experiments, besides the afore-mentionedBac. mesentericus, Ps. fluorescens was also employed. The results of theseexperiments are given in Table 28.

Table 28
Overgrowth of clay by bacteria and actinomycetes
(in cm per 24 hours at 25i C)

Microorganisms

Sterile clay samples from Experimental Station at Chashinkovo

Nonsterile clay samples from Experimental Station at Chashinkovo

Sterile clay samples from Lenin Hills

Nonsterile clay samples from Lenin Hills

Sterile clay samples from Demitrov region

Nonsterile clay samples from Demitrov region

Bact. prodigiosum

1.0

2.5

1.5

2.0

1.0

2.0

Bact. coli

1.0

1.5

1.5

1.0

1.0

1.5

Ps. aurantiaca

1.5

2.0

1.5

2.5

1.5

2.0

Ps. flourescens

2.0

--

1.0

--

1.0

--

Az. chroococcum

1.0

1.0

--

--

1.2

1.0

Bac. subtilis

1.0

--

1.5

--

--

--

Bac. mesentericus

2.5

--

2.5

--

2.0

--

Bac. mycoides

0.5

0.5

0

0

0.5

0.5

A. violaceus

1.8

2.0

--

--

2.5

4.0

A. coelicolor

1.5

2.5

1.5

2.0

--

--

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

  The microbial cells do not move through small pores of the naturaland artificial substrates mechanically, or under the action of external pressure,as they do under filtration, but they move as a result of overgrowth. Dead cellsdo 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 passthrough small pores and capillaries. The optimal temperature for the growth and multiplicationof bacteria Is, at the same time, the most favorable for their passage through smallpores. At a temperature of 5-7° C Bact. coli and Bact. prodigiosummultiply slowly and pass through a Chamberlain candle in 60-80 hours. At a temperatureof 25° C this period is shortened to 20-30 hours.

  The motility of microbes in clay is increased upon introduction oforganic 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 precedingexperiment, and, at some distance from it, an elongated groove is dug. Bacteria areplaced in the central well (aqueous suspension), and nutrient substances (those listedabove) in the groove. It was noted that microbial cultures of Bac. mycoides, Bact.prodigiosum, Ps. aurantiaca, and A. coelicolor moved in the directionof 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 ofthe 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 moved2.0 cm in the control as compared with 3.0-4.0 cm in the experiment. The movementof actinomycetes in the presence of organic substances was by 1.0-1.5 cm greaterthan in the control. It should also be pointed out that the degree of permeabilityof small porous substrates also depends on a great number of external factors, suchas the pH of the medium, aeration, and the composition of the soil solution. Anythingthat favors the growth and proliferation of organisms also enhances the overgrowthof the substrate.

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

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

 

The morphology of microbes in soil

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

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

  These problems are very little dealt with in the literature. If ourknowledge 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 accordingto Vinogradskii, or overgrown glass according to Cholodny, not infrequently, theusual forms of bacteria, actinomycetes, fungi, protozoa and others were seen.

  Consequently, the cells of microorganisms in the soil are of the sameform 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 theycontain 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 nutrientsas sugars and meat-peptone broth. Subsequent inoculation on agar media revealed hundredsof millions of bacterial cells in the analyzed sample. An enormous number of bacteriawas 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 shouldhave been able to detect them in the microscopic field (lens 90, ocular 10) in hundredsand thousands, while in reality their number did not exceed 30-50 and more oftenthere were only 10-20 cells*. (* The determination of the number of the bacteriawas carried out as follows: a 4 cm2 square was drawn on a slide, One dropof soil suspension (0.05 ml) was uniformly spread an the square. The smear was air-driedand stained with fluorochrome dyes. It was studied under the microscope with lens90, 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 = 22,686.5µ2. Since there are 100,000,000 µ2 in l cm, thenin the given area there are 4,402 fields.)

  Two to three days after the introduction of MPB into the soil, a largenumber of cells in the form of rods (150-200 in a microscopic field) could be seenin smears and in the glasses of overgrowth. These cells are mostly very small andapparently belong to the group of nonsporiferous bacteria of the genus Bacteriumand Pseudomonas. Not infrequently, cells of the mycobacterium type are encountered,larger cells of the sporiferous bacteria are very rarely found. As a rule, the latterare 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 theglasses of overgrowth. At the same time hundreds of millions of bacteria can be detectedupon inoculation on nutrient media. Consequently, no less than 100 cells ought tohave 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, wereintroduced into the soil. The amount of the bacteria introduced was 100-1,000 millioncells 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 nutrientmedia. Azotobacter was detectable for 3-6 days upon inoculation in artificialmedia; in smears the cells of Azotobacter were found in larger numbers, butthey were all dead. They did not grow in nutrient agar and did not change their formfor long periods of time, as If they were in a state of fixation. Their protoplasmbecame less dense and the disappearance of the reserve food was sometimes observed.

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

  We have studied the rhizosphere of pea and corn, grown in specialcontainers filled with soil or quartz sand. When the plants grew in the sand allthe cellular elements of bacteria, fungi and actinomycetes growing around the plantroots were clearly seen in the imprints on the glass. Bacterial cells present nearthe roots and also on the roots and on the root hairs were of the same form and sizeas those seen after growth in artificial media. Cells of nonsporiferous and sporiferousbacteria, mycobacteria, proactinomycetes and actinomycetes were clearly seen (Figure58). 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 smallcolonies of cells of coccoid form were infrequently observed. Frequently threadsof actinomycetes and fungi were seen. Inoculation of this rhizosphere soil by themethod of sterile dilution revealed hundreds of millions of cells. Analogous analysisof 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 intothe soil was observed by Dianova and Voroshilova (1925). Chudiakov (1926) explainedthis 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, furthermorethis process is of quite a different character.

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

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

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

  Minute corpuscles in the form of debris can always be detected insmaller or larger numbers during the examination of soil smears. These corpusclesstrongly adsorb dyes. We assume that this granular mass consists mainly of soil colloidsand partly of decomposition products of microorganisms. It has been proven experimentallythat they contain cell gonidia. So, if the smear or the imprint on the glass as wellas the glasses of overgrowth, are covered with a thin layer of nutrient agar (orbetter 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 ofwell-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 protoplasmbecomes granulated, lightens and after the dissolution of the membrane, disappearsaltogether. Small granules and cell debris remain. Other cells begin to divide butdo not elongate and the daughter cells in their turn divide without correspondinggrowth. As a result, minute cell elements are formed in the form of small granulesor corpuscles.

  Such cell division was observed in the sporiferous bacteria, Bac.megatherium, Bac. mesentericus and Bac. subtilis, in mycobacteria, incertain strains of nonsporiferous bacteria of the genus Pseudomonas and inBacterium, in root-nodule bacteria (Rhizob. trifolii) and in Azotobacter.In the latter, the decrease in size of the cell due to cell division without concomitantcell growth is quite often observed (Figure 59). Great changes in the cellular elementsare 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. Infavorable conditions, on artificial media, they grow, giving normal offspring.

  We observed transformations of bacterial cells during the decompositionof plant residues (roots or parts growing on the surface). At first the bacteriagrow together with other microbes in the usual rodlike forms. After a certain periodof time, when the vegetative residues have to a certain extent decomposed, the bacteriadegenerate 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, bacteriahave different forms of existence. In addition to those forms, which, from our pointof view, are the ordinary forms, they also most frequently exist in the form of minutecellular elements, as coccoid cells of decreased size or In the form of small gonidialcorpuscles. These forms lead an independent existence for an unlimited period oftime. Only under special conditions (excessive nutrition, etc) do they assume a rodlikeform and a size characteristic of each species.

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

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

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

  The process of fragmentation of threads into rodlike and coccoid cellscan also be induced in the actinomycetes on nutrient media, if they are grown withconstant 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 sharpchanges and transformations of individual species of this group of microorganismscannot be precluded.

 

Ecological and geographical distribution of microorganisms in soils

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

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

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

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

  A great number of works are devoted to the distribution of Azotobacterin the soil. Attempts were made to determine some regularities of the ecologicalorder 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. mycoidesin soils of the USSR, the author gives numerous data collected by him during manyyears. According to his data, the microorganisms are distributed in the same wayas the higher plants in strict relation with the geographical location. Certain speciesexist in the North and others in the South. According to the author, microbes changetheir biological properties according to the geographical conditions.

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

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

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

  Another difficulty in the compilation of a geographical chart of microorganismslies in the fact that individual bacterial species are distributed in the soil, notdiffusely, but in separate colonies and foci and, in addition, they often grow andcan be detected only in certain seasons.

  The number and the size of the foci or individual concentrations ofmicrobes also depend on the kind and state of the soil, as well as upon the seasonand other conditions. Sometimes, analyses have to be carried out in many dozens oreven hundreds of samples, in order to be able to detect the presence of this or anothermicrobe, and the degree of its growth. This explains the variability of the dataon the distribution of Azotobacter or other bacterial species in one and thesame soil.

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

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

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

  When considering the microorganisms we cannot say whether there areamong them endemics and stenochores--adapted for growth in certain restricted geographicalzones. Microbes are known which live in hot springs, the temperature of which exceeds60-70° C. Microbes maybe encountered in a milieu saturated with H2S,CH4, and other substances. These microorganisms are endemics, restrictedto the ecological zone. No data are available in microbiology on the existence ofa 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 thrivesin 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. mesentericuscan be encountered in all geographical zones. The root-nodule bacteria of astragalsand clover are found in the soils of Central Asia, Caucasus, Crimea, the moderatebelt of the RSFSR and also in the soil of the Kola Peninsula, the islands of theArctic Ocean, Severnaya Zemlya, Franz Joseph Land and others. (Kriss, Korenyako andMigulina, 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 Peninsulaand Moscow Oblast',

  For a long time the yeast Schizosaccharomyces octosporus wasconsidered in the mycological literature to represent the typical population of thesouthern 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. globisporusand A. streptomycini are found in the soil of the Kola Peninsula, in the MoscowOblast' and in the Caucasus, in the Crimea and in Central Asia. The violet actinomyceteA. violaceus and the blue A. coelicolor live in soils of the Vakhshvalley, Crimea, Caucasus, Moscow Oblast', Igarka, Astrakhan' and Leningrad. Accordingto 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 speciesare cosmopolites.

  Certain representatives of physiological groups of microorganismsare even more widely distributed. Thermophiles and psychrophiles can be detectedin considerable numbers everywhere, In the Far North, in the tropics, on mountainsummits 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 theseorganisms are cosmopolites. However, a more detailed study shows this to be wrong.The afore-mentioned organisms do not represent individual species but heterogenousgroups. In each group there are species sharply differing from each other. Some ofthem are widely distributed, others restricted to certain localities.

  In medical microbiology pathogenic bacteria are known, whose distributionis restricted to some countries. Among the saprophytes there are forms, some of whichare 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 conditionsare, to a certain degree, determined by the geographical locality; only through theseconditions, may the locality affect the biology of the living organisms. Consequently,,the regularity of microbial distribution is determined by ecological factors andnot by geographical locality as such.

  Different external factors will predominate in each place of bacterialhabitation. 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 factorwill also predominate with the altitude.

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

  The temperature as an ecological factor effects the total number ofpsychrophiles and thermophiles. Investigations show, that thermophiles are encounteredin 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 JosephLand, 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 increases10-50 times.

  It should be noted that the bulk of the microflora of northern andsouthern soils consists of mesophiles, growing within the temperature range of 3-5to 35° C. We have carried out special experiments on the comparison of the microfloraof soils of the northern, southern and moderate belts. The soils were inoculatedinto 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.

Table 29
The number of microorganisms, growing at various temperatures
(in thousands/ gram)

The soil taken from

4° C

10° C

20° C

25° C

30° C

37° C

42°C

Severnaya Zemlya

3,100

3,300

29,800

3,000

2,200

60

0.5

Franz Joseph Land

2,900

2,500

30,000

2,400

2,100

30

0.1

Kola Peninsula, cultivated

1,900

1,70

1,300

1,800

1,800

50

0.1

Moscow Oblast', cultivated

2,400

2,800

3,100

2,800

3,400

100

2.0

The southern shore of Crimea, under vines

3,800

4,600

6,700

5,400

5,800

250

10.0

Araseuli, Caucasus, under tea bushes

1,800

1,900

2,500

2,000

2,200

250

15.0

 

  Most bacteria have a different minimal temperature of growth, dependingon the place of isolation. Cultures isolated from the soil of Franz Joseph Land havea minimum at 3° C and their growth starts after 10-12 days; those from the soilof Moscow district start to grow after 15-17 days and bacteria from the Crimea andCaucasus start to grow after 18-20 days at the same temperature. The maximum temperatureof bacterial growth is even more sharply pronounced. Most bacteria from the soilsof Franz Joseph Land and Severnaya Zemlya cease to grow at 30-32° C. At a highertemperature 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 soilwhich can grow at 32° C. There are many forms that can grow at 42° C. Inthe southern soils of Crimea and Caucasus the number of thermophiles is even higher.

  The given data show that in all geographical localities the mesophilesprevail. They are the forms which are of the greatest importance in the processestaking part in the soil.

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

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

  In dry soils of the southern deserts and steppes the lack of moisturecreates special conditions for the growth and formation of xerophytes. These formsare frequently found among mycobacteria and actinomycetes. According to our observationsin the Volga steppes, in the Kara Kum desert and in other. dry regions, mycobacteriaprevail in periods of low humidity.

  Saline soils are inhabited by halophytes, organisms which can toleratesalt, including many groups of bacteria, mycobacteria and actinomycetes. Sushkina(1949) described a halophyte form of Azotobacter. The halophils can be encounteredamong the sporiferous and nonsporiferous bacteria. They can be isolated from normalnonsaline 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 theyreach tens and hundreds per gram.

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

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

  One of the main reasons for our ignorance of the ecological geographicaldistribution of microorganisms is the difficulty in the determination of the individualspecies. 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 variationsdiffering 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 soilin various forms.

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

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

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

  A reverse phenomenon takes place in some other species. Differentvariants may have the same external appearance and the differences in their physiologicaland biochemical properties are not easy to determine. It is very difficult to distinguishsuch variants. Such forms are encountered in Az. chroococcum and in many speciesof 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 culturesrepresenting separate taxonomic entities, strains, forms, varieties or even species("sufficient" to justify further taxonomic division).

  The genera Bacterium, Pseudomonas and others are even morediverse. The inadequacy of the methods of differentiation of these organisms doesnot enable us to detect this diversity. Bacteria taxonomically belonging to a singlespecies in reality represent whole groups, which in turn should be subdivided intoseparate taxonomic entities.

  Differentiation of such species or groups in actinomycetes can beaccomplished. By employing the method based on the specificity of antagonism, wewere able to disclose the complexity of some species of monolithic taxonomic entitieswhich were designated as species. For example, the former species A. globisporusis now divided into approximately 10 species and A. coeliccolor into 7-8 sufficientlyeasily differentiated species, etc.

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

  In what manner are all these forms distributed in the soil? Are theydistributed according to geographical zones, or according to ecological conditions?Investigations show that the different forms, variants and even species can be frequentlydetected 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 whichdiffer from one another by their cultural, physiological and biological properties.Among the 150 cultures isolated from the soils of the experimental fields of theAcademy of Agriculture im. Timiryazev and from the soil of the Experimental Stationof 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 strainsof root-nodule bacteria of lucerne were isolated from the soil of two regions ofAzerbaijan and studied. Among them more than 50 variants were found. These variantsdiffered sharply from one another. Strains which differed from each other even more,were detected among the root-nodule bacteria of Onobrychis isolated from the verysame sector. Different forms and variants of Az. chroococcum can be foundin 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 Crimeaand In the Caucasus (author's observations). Petrenko (1953) detected a large numberof different forms of Azotobacter, in podsolic soils near Moscow.

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

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

  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 microorganismto grow. In chestnut soils of the Trans-Volga region Azotobacter cannot bedetected, but it appears there immediately after the soils have been irrigated. Fertilizationof soils, especially with organic fertilizers, sharply increases the growth of thisorganism in the podsol soils.

  As already pointed out, the better the soil is cultivated, the moremicroorganisms it contains and the higher its fertility. With cultivation, the numberof 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 manyothers also increase in number. It is difficult to say which of those microorganismsis the best indicator of a cultivated soil.

  Moshustin (1945a, 1947) suggested that the thermophilic bacteria beused 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, bacteriaas well as actinomycetes, are natural inhabitants of the soil. Their numbers increasewith the degree of cultivation of the soil. The other organisms proliferate at thesame time. In some instances the thermophiles increase under more intense cultivationof the soil and sometimes other groups of bacteria and actinomycetes predominate.

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

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

  Thus, in order to draw up a chart of the distribution of microorganismsin 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 arenot available at present and those which are, cover restricted areas only. An immenseand painstaking work is called for, a work which can only be performed by a largeteam of microbiologists, working in different institutions but according to one planand using the same methods.



HOME      AG LIBRARY      GO TO PART III