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Effect of Soil Microorganisms on Plants
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
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.
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