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Part IV, continued: 

  Investigations have shown that plants not only determine the microflora of the soil during their growth but also determine them by means of their dead residues, especially those of roots. It was established that these residues, depending on the species of plant or, more accurately, on their chemical composition, are decomposed by various forms of microbes. The qualitative composition of the microflora of the decaying roots of wheat and clover, cotton and lucerne, differ considerably.

  We performed microbiological studies of the root flora and of the flora of the area around roots which were rotting in the soil after the harvesting of crops. Studies have shown that around the roots of wheat, corn, sunflower, soybean, and other plants, the number of microorganisms is considerably higher than in the soil outside the root zone. If by ordinary calculation (plating on an agar medium) in the soil outside the root zone one finds four to eight million bacteria in one gram, then around decaying roots there are 20 to 140 million. The number of bacteria varies according to the degree of the decomposition of the root tissue.

  The group composition of the microflora of decaying roots is given in Table 85, which shows that the increase in the total number of microorganisms paralleled the multiplication of the cellulose bacteria.

  Actinomycetes, mycobacteria, and coccoid forms appeared somewhat later, after the development of the nonsporiferous bacteria. Coccoid forms are basically mycobacteria, actinomycetes. and, to a certain degree, mycococci, i.e., organisms belonging to the group of actinomycetes.

  Our subsequent studies were made with root residues which were introduced into the soil. We studied the microflora of the decaying roots of lucerne, clover, wheat, corn, and Euagropyrum, introduced in podsol soil in glass vessels.

  At the same time, the microflora of a root-mass compost was studied under the conditions of a laboratory experiment. The results are given in Table 86.

  In the table, data are given on a number of microbes during the initial stage of root decay. During the subsequent stages of decomposition, the quantitative ratios of microorganisms changed; the number of actinomycetes increased considerably, while the number of sporiferous and nonsporiferous bacteria and fungi decreased. In a half-decayed mass of roots, actinomycetes often cover the root particles with a white coating of aerial mycelium.

  Nonsporiferous bacteria prevail during all stages of root decay, but their species composition varies. The number of cellulose bacteria during the root decay of various plants differs. Filter paper spread on a soil plate containing the roots of clover or lucerne decomposes more quickly and more effectively than on soil containing the roots of wheat. In the former case, 500-700 eroded spots were counted on the paper and in the latter case, only 130-270 spots were found. A compost of the roots of Euagropyrum contains fungi which are absent in the decaying roots of clover, and vice verse. In some cases, there is a mass multiplication of mycolytic bacteria and, in other cases, these bacteria are scarce or not detected at all (Krasil'nikov and Nikitina, 1945).

  Bodily (1944) introduced residues of clover and wheat straw into the soil and he observed that in the presence of clover the number of microorganisms in soil was greater than in the presence of wheat straw.

  Other plant residues, in addition to roots, possess, to a certain degree, a determining effect on the composition of the soil microflora. Birch leaves decompose differently and possess a different microflora than leaves of aspen and oak. The composition of the microflora of Euagropyrum straw and wheat straw also differ. Consequently, all the plant residues of the roots, stems and leaves upon decomposition, enhance the growth of different species of microorganisms in the soil.

  Al'bitskaya (1954) obtained data on fungi from her study of the decomposition of plant residues of forest and steppe vegetation. According to these data, the roots of steppe vegetation are mainly decomposed by fungi of the genera Penicillium, Cephalosporium , Fusarium, and also, to a limited extent, by members of the genus Mucorales. Roots of oak are decomposed by fungi of the genus Trichoderma--T. lignorium, T. koningii, and rarely, by members of the genus Penicillium.

  Plant residues of both steppe and forest vegetation are more intensively decomposed after being inoculated by a mixture of the natural microflora where, in these cases, the greatest loss in water-soluble organic substances is observed, which indicates a most complete decomposition of the residues. Upon the decomposition of steppe vegetation, there is a greater CO₂ evolution and a decrease in water-soluble substances and in lignin. Upon the decomposition of the roots and leaves of oak, the water-soluble substances are depleted in carbon, and upon the decompo sition of steppe vegetation, their car bon content increases.

  Thus, plants, while alive, differentiate, select, and accumulate certain compounds. In other words, the vegetative cover, as a whole, is a powerful determining factor in the microbial biocoenoses of soils. In the zone of the root system, only those organisms which assimilate the root excretions of the plant in question more quickly, can develop, supplanting other, less well adapted, species.

  In different plants, the dominating microbial forms differ in their systematic position as well as biologically. It may be said that each species of plant, or group of closely related species, concentrates a more or less specific microflora.

  Unfortunately we are not yet in the position to determine this specificity in a precise way. Our methods of recognizing and classifying microbes, especially bacteria, are far from being completely developed. We cannot exactly say in what manner the nonsporiferous bacteria, which dominate in the rhizosphere of wheat differ from the bacteria in the rhizosphere of clover, oats or potatoes. By their appearance, cell size, motility, colony structure, and nature of growth on media, they do not differ in the majority of cases, nor do they differ in their generally accepted physiological properties.

  Only a more thorough study of the biochemical activity of microorganisms will enable us to differentiate between them. However, such a method of study has not yet found wide use in laboratory investigations of rhizosphere microflora.

  The selective action of the vegetative cover may be directed toward the selection not only of useful, but also of harmful microflora. Under unfavorable conditions, when agrotechnical rules are not observed, with an incorrect choice of crop rotation, the fields are contaminated by phytopathogenic bacteria and fungi and other harmful microbes--weeds. Especially, after the prolonged repeated cultivation of plants on the same field in monocultures, one observes this effect. The accumulation of an undesirable microflora under monocultures is most often caused by the insufficient growth of microbial antagonists in the rhizosphere, which are characteristic of the given plant under conditions of normal growth.

  Many outbursts of epiphytotic diseases, as for example fusarial infections of cereals, cotton, saplings of woody plants, as well as cotton wilt are, in our opinion, caused by the above phenomenon. This was proved by microbiological studies made of cotton fields which were afflicted by the fungi Verticillium dahliae and Fusarium vasinfectum. These fungi are removed as soon as mycolytic bacteria begin to grow in the soil. These bacteria, as was shown above, grow under lucerne and certain grass mixtures.

  In agricultural practice from time immemorial, crop rotation has been used as one of the methods of increasing crop production. It was empirically found that with certain crop rotations, not only crop production increased, but disease decreased. It is known that lucerne ameliorates the soil in cotton farms and inhibits the growth of the organisms causing cotton diseases. On this basis, one can select plants for rational crop rotation.

  Noting the great influence of the vegetative cover on the formation of microbial biocoenoses in soil, one should not forget the importance of the soil itself as a substrate and the effect of the activity of man on external conditions. The physicochemical state of soil determines to a great extent the direction of the microbiological processes, and to a similar extent the development of different microbial species. The distribution of Azotobacter in soil not only depends on the plants. on their root excretions and decomposition products, but also on soil acidity and the presence of phosphorus, calcium, molybdenum, and other nutrient elements.

  The cultivation of soil, the liming of soils, the use of fertilizers and other factors favor the growth and accumulation of Azotobacter. In arid regions the growth and accumulation of Azotobacter in soils depends to a considerable degree on irrigation.

  In the foregoing chapter, the considerable influence of the microbial population of the soil, and the accumulation of individual species and groups of microorganisms in the root zone, was shown. The importance of root microflora for the life of plants has been studied only a little and the information available on the action of different microbes on the growth of plants is meager. We will not dwell here on the activity of root-nodule bacteria, Azotobacter, and mycorhizal fungi, since data on this subject are abundant in scientific literature. In this chapter, data are given only on those organisms which exert a beneficial or harmful effect on plants, due to the products of their metabolism; these are microbial antagonists, activators, inhibitors, etc.

  It was noted above that certain soil microorganisms are capable of producing various biotic substances--vitamins, auxins, amino acids, and other biocatalysts. Such microorganisms activate the biological processes and, therefore, we named them microbial activators.

  There is much data in the literature on the positive effect of pure cultures of bacteria, fungi, and actinomycetes on the growth and development of plants. Microbial activators increase the percentage of germinating seeds, enhance the growth of the young plants, and often change the nature of the biochemical processes.

  Already toward the end of the last century, Geier (1882), and later Zimmermann (1902), described the bacteria living in the tissues of plants which had a certain activating effect on their growth. Such bacteria could form special nodules in the leaves of subtropical and tropical plants.

  According to their systematic position, these bacteria differ from each other. In members of the genus Ardisia Thunb., the nodules in the leaf tissues are formed by nonsporiferous bacteria of the genera Bacterium and Pseudomonus. In Pavetta L., Chomelia L,, Psychotria L., and certain other genera, mycobacteria were isolated from the nodules, in Dioscorea L., and others, bacteria of the Rhizobium type were isolated (Krasil'nikov, 1940 a, b).

  Miehe (1911, 1918) studied in detail the bacteria which are members of the genera Ardisia Thunb. and Pavetta L. According to his data, they formed special substances which cause the stimulation of the tissues (Reizwirkung). They do not fix nitrogen.

  Jongh (1938) found that Ardisia does not grow or grows poorly without bacterial symbionts, and does not bloom nor bear fruit. When bacteria were introduced into the tissue of the plant, its growth and development improved drastically, the growth of branches was enhanced, leaves acquired normal form, and flowers and fruits appeared.

  The role of the symbionts of the root-nodule bacteria group is widely known. Forming nodules on the roots of leguminous plants and on certain nonleguminous ones, under certain conditions they considerably improve the growth of plants and increase crop yields.

  The biological role of root-nodule bacteria for plants is widely known. However, the mechanism of the action of these organisms is still obscure. It is assumed that root-nodule bacteria fix molecular nitrogen and supply it to the host plant. There is no clear-cut experimental data to support this assumption.

  There are reasons to believe that root-nodule bacteria, as well as the bacteria from the nodules on the leaves of the above-mentioned plants, act favorably through their metabolites. According to our data, leguminous plants, in symbiosis with the nodule hacteria, fix molecular nitrogen for themselves from the air. The bacteria, due to their metabolic products, act as biocatalysts, activating the nitrogen-fixing ability (Krasil'nikov and Korenyako, 1946a).

  The positive action of mycorhizal fungi has already been mentioned. These fungi are widespread in nature. One view has been expressed that all plants have mycorhizae, but differ as to the nature of the co-habitation. In some plants the mycorhiza is endotrophic and in others ectotraphic. In the former, the fungal hyphae grow almost exclusively in the root tissues and only a few extend into the soil, outside the root, The endotrophic mycorhiza, in its turn, includes two types of mycorhizae the phycomycetal and vesicular type, wore often encountered in grassy and woody plants, and the orchid mycorhiza found in the plants of the orchid family These two types of mycorhiza differ in the nature of the structure and development of their mycelial hyphae. In phycomycetal mycorhizas, the hyphae are not septate and often form characteristic swellings in the root tissues--vesiculae. The mycelium in the orchid mycorhiza is septate; the hyphae form characteristic entanglements only within the root cello. As a rule, they do not have vesicles.

  In the endotrophic mycorhiza of both types, the hyphae develop only in the cortex of roots in the intercellular space, or penetrate into the cells. The mycelia of the fungus do not penetrate into the central part of the root.

  Ectotrophic mycorhiza is characterized by the growth of mycelial hyphae on the surface of the root tips, which surrounds them with a thick and quite dense cover. From this cover the hyphae extend into the soil. There are no root hairs on this part of the root. A small part of the hyphae penetrates inside the root, but not very deeply, their growth being usually limited to the intercellular space of the epidermis, where the hyphae interweave, forming he dense Hartig net. The hyphae seldom penetrate into the upper two to three layers of the cortical cells. The hyphae do not penetrate the cortical cells, and, in the few cases where this does happen, they soon die inside the cells. Entotrophic mycorhizae are most often encounter ed among woody coniferous and leaf-bearing plants.

  Between the endotrophic and ectotrophic mycorhizae, there are intermediate formations--the ecto-endotrophic mycorhizas.

  Some authors (Lobanov, 1953) are of the opinion that an absolutely ectotrophic mycorhiza does not exist at all. They maintain that in woody plants, especially during the early stages of their development, there is always an endotrophic mycorhiza. Only later the external cover on the root tip develops.

  The systematic distribution of the mycorhiza fungi varies. Fungi-forming vesicular mycorhiza probably belong to Phycomycetes of the Endogenaceae family, The endotrophic mycorhizas in orchids are formed by the Fungi Imperfecti of the genus Rhizoctonia, while in certain orchids, the mycorhiza is ferried by higher fungi, Basidiomycetes with well-developed fruiting bodies--Armillaria mellea, Xerotus javanicus, and Marasmius coniatus.

  Ectotrophic and endotrophic mycorhizae of woody plants are mainly formed by mushrooms, most often by the imperfect fungi of the genus Phoma. There are mycorhiza-forming fungi, among the Ascomycetes and other groups of fungi. For instance, in beech, 12 different species of fungi have been described as participants in the mycorhizae in pine 17 species were described; and, in spruce, up to nine species, etc (Kursanov, 1940; Yachevskii, 1933; Kelly, 1952; Magru, 1949; Lilly and Barnett, 1953, Goimann, 1954, Lobanov, 1953 and others).

  These data clearly show that there is no strict specificity among mycorhizal fungi, as in root-nodule bacteria. The first to observe the positive effect of mycorhizal fungi on the growth of plants was Kamenskil (1880) and after him, Voronin (1886), Vysotskii (1902), and others. They all looked upon mycorhizal fungi as symbionts, which exert a great influence on the growth of plants. Baraney (1940) presents extensive data confirming this point of view. One of his tables is given below (Table 87).

  The essence of the action of fungi consists in supplying the plants with nitrogenous and carbonaceous elements of nutrition in some cases and, in others, in the supply of auxiliary nutrients or biotic substances, and more correctly with both. There is a great deal of data in the literature on the significance of mycorhizal fungi in the nutrition of plants, which was already mentioned In the previous chapters of this work. Recently, by the use of direct experiments with labeled atoms, it was shown that mycorhizal fungi take up find transmit various nutrient elements.

  Kramer and Wilbur (1949) and Mellin and Nilson (1950, 1952) have shown that fungi transmit P³² and N¹⁵ from the external solution into the tissues of the roots and stems of pine (Pinus taeda L., P. resinosa, P. silvestris L.). Morrison (1954) found that in the presence of the mycorhizas, there in enhanced transfer of P³², not only to the roots and atoms of Pinus Rediata, but also to the leaves. Herley and MacCready (1950, 1952) have shown that mycorhizal fungi take up labeled phosphorus, accumulating up to 90% in their mycelia.

  The data on studies made with labeled atoms does not disclose the nature of the compounds by way of which the labeled phosphorus and nitrogen are transmitted to the mycorhizal fungi, one must assume that these elements when entering the cell of the fungus, take part in the general process of building its substance in the form of one of the organic compounds of metabolic products, The labeled elements are released from the cell as metabolites which enter into the medium and, from there, into the roots and green parts of the host plant. Such it process was demonstrated in Shavlovskii's experiment with rhizosphere bacteria (see above).

  The free-living soil bacteria also have a considerable effect on plants. Clark and Roller (1931) studied the action of pure cultures of the following bacteria on the growth of duckweed: Bact. coli, Clostridium sporogenes, Clastrid welchii, Ps. fluorescens liquefaciens, Bact. aerogenes, Staph. aureus, Bac. subtilis, Bact. prodigiosum etc. Some of these bacteria stimulated the growth of buckwheat, while others had no visible effect on it.

  Kozlowski (1935) observed the action of pure bacteria cultures on the growth of barley and apples, The tested cultures of the sporferous bacteria, Bac. cereus, Bac. mycoides, and Bac. subtills and of the nonsporiferous bacteria, Bact. denitrificans, Bact. putidium, and Ps. pyocyanea and also cultures of fungi. The sporiferous bacteria had no effect on the growth of plants. Of the nonsporiferous bacteria, Ps. pyocyanea and Bact. putidum inhibited their growth, while Bact. denitrificans, stimulated the growth of barley, but not that of apples.

  We tested 130 different bacteria, mycobacteria, and actinomycetes isolated from various soils. Among them were the following; 32 strains of Azotobacter, 33 strains of root-nodule bacteria, 40 of Pseudomonas, 10 of Bacterium, four of mycobacteria, six of actinomycetes, three of Bac. mycoides, and two of Bac. subtilis. Cultures of these organisms and products of their metabolism were added to the medium in which wheat seedlings were grown in one series of experiments, and to that of isolated roots of peas and wheat in another series of experiments.

  Isolated roots are a convenient object for the study of the requirements of biotic substances, since they are incapable of the independent synthesis of the whole gamut of these substances.

  We grew isolated roots of peas, wheat, rye and other plants on Bonner's synthetic medium of the following composition:

Distilled water 1,000 ml
Ca (N0₃) ₂ ^. 4H₂ O, 0.23 g
MgSO₄ ^. 7H₂O, 0.36 g
KNO_3,,0.81 g
KCl, 0.65g
KH₂PO₄,0.12 g
Iron, Traces
Saccharose, 20 g

  Extracts and filtrates of bacterial cultures were added in various amounts, as auxiliary substances. The bacterial cultures were grown in liquid media and filtered with bacterial filters. The results are given in Table 88.

  As can be seen from the table, a large increase in the length of the roots was observed in the presence of filtrates of Azotobacter, root-nodule bacteria, and bacteria of the genera Pseudomonas and Bacterium (Figure 82) Metabolic products of certain actinomycetes were quite active. Sporiferous bacteria often showed a negative action, inhibiting the growth of roots.

Figure 82. Effect of products of bacterial metabolism on the growth of isolated roots of peas:

1--metabolites of Ps. fluorescens ; 2--metabolites of Ps. aurantiaca; 3-control (no metabolites added).

  In the same microbial group and even in the same species, different strains showed different effects on the roots. For example, among cultures of Ps. fluorescens, strain No 4 strongly activates the growth of wheat roots, while strain No 15 only slightly activates or does not activate these roots at all; strainNo 15 of Ps. nonfluorescens is inactive, while strains Nos 5 and 10 are active; the root-nodule bacteria of lucerne did not promote the growth of these roots and even suppressed them, while the root-nodule bacteria of peas and especially those of beans greatly enhanced root growth. The same was observed in all other groups of organisms.

  The roots of different plants reacted differently to the action of filtrates of the same culture. The roots of wheat reacted more intensively than the roots of peas to the metabolites of Ps. nonfluorescens, strain 10, while with the filtrate of strain 12, the picture was reversed.

  Filtrates of microbial cultures show a positive effect only when used in small amounts. When the amounts are large, their effect on the growth of isolated roots is negative. The roots do not grow or grow very poorly, deviating from the normal, they thicken, swell, do not branch, become brown too soon, and die.

  The metabolites of many organisms in small concentrations strongly suppress the growth of roots. Such organisms which produce toxic substances are found among various groups of microorganisms but they are especially numerous among the sporiferous bacteria. This group of bacteria is in general the most toxic in relation to plants and many microbes (see below).

  Similar data were obtained in experiments with plant seedlings. The filtrates of certain microorganisms noticeably activated the germination of seeds, while the filtrates of others had an inhibitory or no effect.

  As in the experiments with isolated roots, filtrates of Azotobacter, nonsporiferous bacteria of the genera Pseudomonas, Rhizobium and Bacterium, and many species of actinomycetes most actively enhanced the growth of seedlings, In the genus Azotobacter, the strains of Az. vinelandii and Az. agile var. jakutiae were active. The strains of Az. chroococcum differed considerably in activity. Some possessed strongly and accentuated activating properties with respect to the growth of wheat, others had only weak activating properties and still others had no effect whatsoever on the growth of plants. Under the influence of certain strains, the suppression of wheat growth was observed.

  We studied the activating effect of bacteria on different plants under field conditions for a period of three years (1945a). The seeds were treated with a culture of bacterial activators and were sown on kolkhoz fields in various regions. Altogether more than 100 experiments were performed, not including those with Azotobacter. The summary of the results is given in Table 89,

  The data in Table 89 show that bacterial activators exert a similar effect on crops as do azotogen*, nitrogen, and other bacterial compounds. In our experiments, Azotobacter was used in the form of peat azotogen. *[*Azotogen--Russian commercial name for azotobacter field-inoculating preparation.] In Table 89 are given only those cases where a positive effect was obtained when Azotobacter was absent from the soil; it had perished during the first few days after its introduction into the soil. Therefore, the effect was caused, not by Azotobacter, but by other microbes.

  Akhromeiko and Shestakova (1954) successfully tested bacteria, which had been isolated from the rhizosphere, on the growth of oak and ash tree seedlings. With oak, there was a 24-34 per cent increase in the increment of dry matter, and with ash a 40 per cent increase. Samtsevich and others (1952) used an Azotobacter culture for the inoculation of oak seedlings in a steppe zone. According to their observations, this microbe increases the percentage of acorn germination and enhances the growth of oak seedlings. Similar results were obtained by Runov and Enikeeva (1955), Mishustin (1950b) Smali (1951) and others.

  Shtern (1940 a, b) tested radiation strains of Azotobacter on oat seedlings, grown in vegetation containers. Certain radiation strains were more active than the initial culture.

  Afrikyan (1954a) studied a large collection (more than 200 strains) of sporiferous bacteria isolated from Armenian soils. The wheat seeds which were inoculated with these cultures were allowed to germinate either in Koch dishes on cotton or in sand in containers. The experiments showed that there are very few activators among the sporiferous bacteria of the Bac. subtilis and Bac. mesentericus group. More often one finds bacterial inhibitors in this group which suppress seed germination and the growth of plants. These data are in agreement with our observations (see below).

  Popova (1954) employed cultures of bacteria isolated from the rhizospere of grape vines for the enhancement of the germination of grape seeds and grape stalks. Certain species of Ps. sinuosa increased the percentage of germinating seeds to 80% while in the control plants, only 10-12% of the seeds germinated by the 45th day. These bacteria also enhanced the growth of seedlings and roots. In the control plants, the buds swelled on the 16th day and, in those treated with bacteria, on the fourth day. The highest activity was shown by Azotobacter chroococcum and the nonsporiferous bacterium Bact. album, strains 2 and 3.

  Pantosh (1955) found that nonsporiferous bacteria of the Pseudomonas and Bacterium groups have an activating effect on the growth of the plant from the rhizosphere of which they were isolated. In experiments performed in vegetation containers, on quartz sand, the inoculation of the seeds with bacteria before sowing increased a crop of wheat by 20-65%above that of the controls, as follows:

  Petrosyan (1956) studied the effect of bacterial activators on leguminous plants, on the formation of their nodules and on their accumulation of nitrogen. The experiments were performed in containers and in plots under field conditions. In the former case, the following results were obtained:

  Similar results were obtained in field experiments.

  The increase in crops due to bacterial activators was as follows: after vetch, 172.6%--control crop of 11.6 kg; after lucerne, 141.3%--control crop of 10.2 kg; after Onobrychis, 150%--control crop of 8.4 kg from one plot.

  In our experiments, we obtained similar results. In vegetation containers certain bacterial activators (Ps. aurantiaca No 1, Pseudomonas No 145, Bacterium sp. No 160 produced an increase in leguminous plants as follows: clover, beans, lucerne and lupine, by 30-80% above that of the control plants. In field experiments the crop increase of these plants after inoculating them with the activators, was 24-30% above the control levels. The number of nodules increased considerably when activators were employed.

  On the roots of one plant, the following number of nodules were found: in control plants without inoculation of bacteria, 8; on plants inoculated with root-nodule bacteria of lucerne, 9-12; and after inoculation of bacterial activators (Ps. aurantiaca), 28. On the roots of beans, the number of nodules was 6. 8 and 16; on the roots of lupine, 0.2, 0.5 and 1.2 (Krasil'nikov and Korenyako, 1945c).

  Bacterial activators stimulate the activity of root-nodule bacteria, enhancing the formation of nodules and, by means of the latter, the growth of the plants. Activators also act positively on leguminous plants without nodule bacteria directly stimulating their growth by their metabolic products.

  In one series of experiments, we employed avirulent strains of the root-nodule bacteria of clover and vetch, obtained experimentally. The seeds were inoculated with cultures of these bacteria and sown in sterile sand in glass containers. Crops were obtained after two months.

Figure 83. Organotropic action of bacteria on lucerne:

Exp. A: a--stimulation of root growth with the simultaneous suppression of the growth of the aerial parts; b--control plants; exp. B: a--stimulation of the growth of aerial parts; b--control plants.

Figure 84. Effect of metabolic products of bacteria on the growth of Phycomyces blakeslseanus:

1--control growth of the fungus in the absence of metabolites; 2--mass formation of zygotes (dark spots) in the presence of metabolites of bacterial stimulators (Bacter. sp. No 2); 3--abundant growth of aerial mycelium with conidia in the presence of metabolites of bacterial activators (Azotobacter chroococcum).

  Similar results were obtained in experiments with vetch. Using avirulent experimental strains of root-nodule bacteria, Nos 1, A, D, and A₁, the crop was considerably greater (123-161%) than when the inoculation was performed with the initial virulent culture (107-115%). In experiments with avirulent bacteria, no nodules were found on the roots of the plants while when the initial culture was used for Inoculation, 9-58 nodules were found on each plant.

  Certain chemically pure substances obtained from cultures of actinomycetes and other fungi, as for instance gibberellins and gibberellin-like substances, activate the growth of plants (Figure 85).

Figure 85. Effect of antibiotics on the growth of corn:

1--seeds treated with antibiotic solution before sowing, 2--control, seeds treated with water.

  Dorosinskii and Lazarev (1949), Dorosinskii (1953), Lazarev and Dorosinskii (1953) grew oats in sterile, well-washed, loamy soil in the presence of bacteria and in their absence.

  The plant crop in the absence of bacteria but with full mineral fertilization was, on the average, 2.6 g; in vessels with bacteria, 7.1 g; in vessels with bacteria, but without a mineral fertilizer, 6.5 g.

  Fomin (1951) grow certain melon cultures, fruit, and wood varieties of plants, treating them with preparations of Azotobacter, Psaudomonas, and "silicate" bacteria. After such treatment, the crops of all these plants increased.

  Certain microorganisms exert an organotropic action on plants: they activate the growth of individual organs or tissues. For instance, Ps. tumefaciens stimulates the multiplication of the cells of the root tissue or stem tissue in tomatoes, carrots, and other plants, as a result of which swellings are formed, There are microbes which, by their metabolic products only, activate to a considerable extent, the growth of roots or serial parts.

  In our investigations, we have observed bacterial cultures which, under the conditions of vegetation experiments (in sand) only stimulated the growth of roots, not affecting the aerial parts at all. In other variations of the experiment some bacteria enhanced the growth of the aerial parts without influencing the root system (Figures 83 and 85). Observations were also made of bacterial cultures which activate the process of fertilization in fungi (Figure 84) and yeasts, In Table 90, data are given on the activity of the substances which stimulate this process.

  Molliard (1903) observed the stimulation of the formation of apothecia in the fungus Ascobolus under the influence of the bacteria within. Sartory (1916) obtained perithecia in aspergilli only in those comes where the fungi grow together with the sporiferous bacillus Bac. mesentericus.

  Stimulation of the copulation of the fungus Phycomyces blakealesanus when grown together with bacteria or their metabolic products (factor Z), extracted from agar-agar, was observed by Robbins and Schmidt (1939, 1945). This "factor", according to their data, is present in the tissues of many plants and is also formed by microbes.

  Nickerson and Thimann (1941, 1943) found that a certain substance among the metabolic products of the fungus Aspergillus niger enhances the copulation of the yeast Zygosaccharomyces. The active principle of this stimulant is soluble in water and 90% ethyl alcohol and consists of two substances: an acid closely related to glutamic acid, and riboflavin, Burnett (1956) caused an increased copulation and the formation of zygotes in mucor fungi by the addition of ß-carotene to the medium.

  In our collection, there are actinomycete-antagonists, the metabolic products of which have a stimulating effect on the fruiting processes in higher plants. One such culture enhanced flowering and fruition in the cotton plant. Under conditions of the experiment in growth containers, plants treated with the native liquid of the given actinomycete had 10-12 bolls, while the control plants had 5-6 bolls on each bush. Correspondingly, the cotton crop approximately doubled in quantity. We treated plants with nonpurified substances. It should be assumed that chemically purified preparations will have an even stronger activating effect on plants.

  In recent years great interest has arisen over gibberellins and, especially, gibberellic acid, as a stimulant to the growth and development of plants. These substances are obtained from the fungus Gibberella fujikoroi (a conidial stage of Fusarium moniliforme). This fungus was first isolated in Japan by Kurozava, in 1926, from the tissues of diseased rice. The rice disease caused by this fungus is quite widespread in Japan, and expresses itself in an extreme elongation of the stems, in the yellowing of leaves, and in the death of the plant. Yabuta isolated the active substance from a culture of the fungus and he found that it stimulated the growth of many plants. Later, this substance was isolated in the crystalline form and studied in greater detail. In England and in America, three substances, gibberellin A₁, gibberellin A₂, and gibberellic acid (gibberellin A₃) were obtained from the culture fluid. The last substance is the most active and is of the greatest interest. It was, therefore, more thoroughly studied and more fully elucidated, Chemically defined, it is a dihydroxylacetone acid, a tetracyclic compound, with the general formula C₁₉H₂₂O₆.

  The activating effect of gibberellic acid expresses itself in extremely small concentrations. Only 0.01 microgram/ml is enough to stimulate the growth of peas; at higher concentrations the stimulation of growth increases. The greatest effect is observed at a concentration of 10 micrograms/ml. According to the data obtained by Brian (1957), the increment in the growth of peas exceeds by 10 times that of the control plants. Its height was 42 cm after treatment with gibberellic acid, while in the control, nontreated plants, it was 7 cm. This stimulating effect was also observed by Brian in experiments with wheat, but to a lesser degree than in the experiments with peas. Gibberellic acid does not show an activating effect on the growth of roots, neither in peas nor in wheat.

  Of special interest is the effect of gibberellic acid on the growth of biennial plants: cabbage, rape, carrots, sugar beets, etc. It is known that these plants give off flower shoots and bear fruit during their second year of life; during their fire, year of growth only a rosette of leaves and roots is formed. The flowering and formation of seeds may also be achieved during the first year of growth, but only after yarovization [a Russian term for vernalization; translator].

  Experiments have shown that these plants form flower shoots and flowers and produce seed during their first year of growth after being treated with gibberellic acid, without previous yarovization, This acid has the same effect as the one obtained by yarovization. The enhancement of flowering and fruition is observed in longday plants. Lang (1956) obtained flowers and fruit on henbane (Hyoscyamus niger) during the first year of its growth. After the introduction of 300 micrograms/ml in the tissue, the plant soon formed flowering, or main shoots, on which flowers developed.

  During the last two or three years, a vast amount of material has accumulated showing the strong activating effect of gibberellic acid on the growth and flowering of various plants. The strongest effect is seen with the long-day plants.

  It should be noted that the stimulating effect of gibberellic acid was so far only obtained under experimental conditions in a hothouse. Under field conditions in soil, there have either been no results, or a weak stimulation of only certain plants has been observed. A small increase of crops, within the range of 11-25 per cent has been observed in meadow grasses.

  In plants which react to gibberellic acids, one often observes that there is a decrease in the amount of chlorophyll, that the leaves have a yellowish tint, that their total nitrogen content decreases, and that in the tobacco leaves the percentage of nicotine decreases and , in rice, a decrease in the amount of sugars. It is assumed that this is caused by a lack of nutrition. With appropriate fertilization this has not been observed.

  The mechanism of the action of gibberellic acid is not clear; however, all the investigators note that it differs from that of auxins. The latter affects the processes of growth in a different way. These substances also differ in their chemical composition.

  The data given in this chapter show that there are species of microorganisms which form very active substances, which stimulate the growth and certain processes and functions in plants. Gibberellic acid is the first metabolic product of microorganisms obtained in a chemically pure form. It should be assumed that in the near future many other substances will be obtained which possess the capacity to activate the growth and development of plants. Similarly, as takes place among microbial antagonists. The stimulating substances of the microbial activators belong to various classes of compounds. Here, a great research study lies ahead in the isolation of these substances and in the study of their nature.

  One must note that among the antibiotic substances and activating compounds, there is often much in common in the way they act on organisms. Many antibiotics show a stimulating effect on the growth of plants and animals; they enhance the increment of live weight of the latter, and sometimes enhance the process of fruition in both higher and lower plants. On the other hand, activating substances quite often possess clearly expressed antimicrobial properties.

  Very little is known on the distribution and growth of microbial activators in soil. At the same time, in daily laboratory practice, one very often encounters these bacteria, We have in mind the auxoautotrophs, which have been mentioned before. These organisms synthesize all the substances necessary for growth and development, and, therefore, grow well on simple synthetic media. For the purpose of their study, we used the following medium:

  There were no vitamins or auxins in the medium.

  The total number of auxoautotrophs growing on this medium was quite large. We counted from several tens of thousands to many hundreds of millions of bacteria in one gram of soil.

  The number of auxoautotrophic bacteria changes, depending on the properties of the soil and climatic conditions. As a rule, their total number is greater in fertile soils than in nonfertile ones. When the auxoautotrophs from the chernozem soils of Moldavia, Crimea, and Kuban are plated on an agar medium, 30 to 150 million appear in one gram. In the turfy podsol soils of the nonchernozem belt of the Moscow and Leningrad Oblast's and of Latvia, the number of these organisms varies within the range of 100,000 to 4.5 million per gram. In the soils of the Kola Peninsula (primary soils, loose calcareous soils, etc) there are 5,000 to 30,000 cells in one gram,

  In cultivated soils their number is greater than in noncultivated soils, and in gardens it is greater than in fields (Table 91).

  Comparing the data on the number of bacteria growing on a synthetic vitamin-free medium and on an ordinary protein medium, one can observe that the number of auxoautotrophs and auxoheterotrophs is almost the same. In humus soil the ratio between these two categories is approximately 1:1. In certain cases, there are even more auxoautotrophs (see above).

  According to Schmidt and Starkey (1951), about 30 per cent of soil bacteria synthesize biotic substances and excrete them into the soil.

  Lochhead and Chase (1943) found from 10-14 per cent of auxoautotrophs among the microflora of soil. These authors divide soil microbes into seven groups, according to their ability to grow on vitamin-free media with the addition of a few auxiliary substances. To the first group belong the microbes growing on media completely devoid of vitamins. In the second group are included organisms growing on a vitamin-free medium with the addition of a few amino acids (cysteine, alanine, proline, asparagine, arginine, leucine, glycine, lysine, etc). To the third group the authors relate those microbes which require for growth certain vitamins: pantothenic and nicotinic acids, thiamine, riboflavin or others. The fourth group includes organisms growing on a medium to which both amino acids and vitamins were added. Those microorganisms growing on a basal vitamin-free medium with an admixture of yeast extract comprise the fifth group.

  A basal vitamin-free medium with an admixture of soil extract reveals the bacteria of the sixth group. This medium, with a small admixture of yeast and soil extracts, is suitable for the bacteria of the seventh group, the most numerous one. Microbes of the first group comprise approximately 10-14 per cent of all soil bacteria; the second group, 10 per cent; the third, 12-14 per cent; the fourth 16-17 per cent; the fifth, 18-20 per cent; the sixth, 3-7 per cent; and finally, the seventh, 40-50 per cent of all soil bacteria. This subdivision of bacteria is quite arbitrary.

  A great number of vitamin-producing bacteria are found in the rhizosphere of plants. According to our calculations, they comprise 40-80 per cent of all soil bacteria and their number varies depending on the species of the plant, the stage of its growth, and upon external conditions. In analyzing the rhizosphere of wheat and clover, growing in the fields of the Moscow district Dolgoprudnoe), we obtained the data given in Table 92. In the root zone of these two plants and in all other plants studied by us, the number of auxoautotrophs was no smaller and often larger than the number of auxoheterotrophs.

  West and Lochhead (1940a, b), and Wallace and Lochhead (1949), in their detailed studies also found that auxoautotrophs prevailed in the rhizosphere of flax and other plants. The same was noted by Katznelson and Richardson (1943), Stolp (1952) and others (Table 93).

  As is well known, the ability of microorganisms to produce biotic substances is, in most cases, not connected with their taxonomic division into groups. Auxoautotrophs and heteroauxotrophs are found among various microbial groups: among sporiferous and nonsporiferous bacteria, micrococci, mycobacteria, actinomycetes, fungi, etc. The largest percentage is encountered among the bacteria of the genus Pseudomonas. According to our calculations, the number of these bacteria reaches 0-60 per cent of all the bacteria growing on an agar medium devoid of vitamins. A smaller, but quite a high percentage (30-40 per cent) is encountered among the genus Bacterium and mycobacteria. Considerably rarer are the auxoautotrophs among sporeforming bacteria of the genus Bacillus (Table 94).

  Oligonitrophiles are active producers of biotic substances. These organisms are widely encountered in soils and their characteristic feature is that they grow abundantly in a vitamin-free and nitrogen-free medium. Evidently, they all belong to the auxoautotrophs.

  Among the actinomycetes one rarely finds cultures which will not grow on Chapek' s synthetic medium. They grow well on a vitamin-free medium.

  Yeasts of the genera Torula, Mycotorula, and some others are widespread in the soil. These organisms are powerful producers of vitamins and various other biotic substances. Therefore, they grow well on mineral, vitamin-free media, forming large slimy or semislimy colonies. Some of them, as, for instance, Torulopsis pulcherrima are widespread in the soil and grow abundantly on the nitrogen-free medium of Ashby, forming large colonies similar to Azotobacter colonies. They are of special interest as activators of plant growth and of the life processes of microorganisms.

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