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Part IV, continued:

  The importance of antibiotic substances formed in the soil, for plants, is determined primarily by the extent to which the former enter the plants via the roots and by their activity within the plant.

  The question of whether antibiotic substances are capable of entering the plants is now answered in the affirmative. Vegetating plants absorb various organic substances through their roots including antibiotics formed by bacteria, actinomycetes and fungi. The absorption by plants of subtilin, gramicidin, pyocyanine, licheniformin, (antibiotics of bacterial origin), as well as streptomycin, globisporin, aureomycin, terramycin, grisein, griseofulvin, etc (substances formed by actinomycetes) has been experimentally established.

  Table 121 shows the degree of entrance of chemically pure antibiotic preparations, from solutions into the plant, via the root.

  Penicillin enters the plant at the fastest rate and in the largest amounts, followed by grisein and streptomycin. Mycetin subtilin and gramicidin enter in small amounts and do not travel high up in the plant.

   Aureomycin, terramycin, globisporin and many other active substances enter readily into the plants.

  Plants absorb from the substrate not only chemically pure preparations but also crude antibiotics, in the form in which they are excreted by their producers. We added a liquid containing antibiotics to a culture solution and grew plants in it. After a certain time one could detect antibiotics in the tissues of the latter (Table 122).

  Antibiotic substances enter plants from solid substrates, directly from soils.

  In our experiments (Krasil'nikov 1951 a; 1952 b, c; 1953 c; 1954 a) we have tested various antibiotics formed by bacteria, fungi and actinomycetes-antagonists. Chemically pure preparations were introduced under adult plants in containers with sand or soil and their concentration was determined after given time intervals on the root tissues and aerial organs. The experiments were performed both under sterile and nonsterile conditions of growth. In Table 123 are given the data from the experiment with the sterile sand substrate. Streptomycin 500 units and grisein (preparation 15), 500 units were introduced per 1 g of substrate.

  As can be seen from the table, antibiotics enter into the plants via the roots in quite large quantities. They are found in roots, stems and leaves for 10 days and more.

  In experiments performed with sterile soil (Moscow area podsol) the same results were obtained. The antibiotics, globisporin, 500 units and grisein, 800 units were introduced per 1 g of substrate. Peas, kidney beans and wheat were grown on this soil. Analyses have shown, that globisporin was preserved in soil for 30 days and grisein for 40 days. During this time they penetrated through the roots and into the plants in smaller or larger quantities (Table 123). The rate of entry of antibiotics from sterile soil was only slightly less than the rate of entry from nutrient solutions or from the sand substrate. After a few hours (6-10) the antibiotic substances could be found in the roots and in the lower parts of the stem.

  Our experiments have shown, that antibiotics are also absorbed by plants from natural nonsterilized soil. The same antibiotics, grisein and globisporin, were introduced into vessels with podsol soil under adult plants (wheat and kidney beans) in doses of 800 units/g. After 24 hours and sometimes even later we detected these antibiotics in the tissues of the roots and aerial organs (Table 124).

  Plants also absorb crude antibiotics from the soil. We tested three native antibiotics formed by the actinomycetes No 290, 287 and strain "B." In the presence of the appropriate organic matter in the soil, these cultures, as is shown above, form 30-120 u/g of antibiotic substances. When we grew peas and wheat in such soil, we could detect the antibiotics in the tissues in small, but sufficient quantities which were large enough to inhibit growth of microbes sensitive to them (Table 125).

  Thus, in pea plants, 2-15 units/g antibiotics were obtained and in wheat somewhat less. In some cases, when there is an abundant growth of antagonists which form large amounts of antibiotics in the soil (100-150 units/g), large quantities of the latter enter the plants-up to 20-30 units/g in the roots and 10-20 units/g in the leaves.

  The zones of growth inhibition of the test organism around pieces of tissues of the experimental plants may be seen in Figure 96. Around the shreds of tissue of the control plants no such growth-inhibition zones are formed.

Figure 96. Entrance of antibiotics from the soil into plant tissues. Zones of lack of growth of bacteria around pieces of tissues:

1--roots; 2 and 3--stems (lower and upper parts); 4--leaves; 5--roots of control plant, grown in soil without antibiotics, the zone is missing.

  It should be noted that plants may not only absorb the antibiotics that exist in the free state from the soil but also those adsorbed by soil particles. It was stated earlier, that a considerable part of the antibiotics are adsorbed immediately after their formation and become tightly fixed. Adsorbed antibiotics cannot be washed,out with water or with a number of organic solvents, even upon prolonged treatment. However, due to the activity of their root systems, the plants, are in the position to sever this, bond between the antibiotics and the soil particles and to desorb and take up the antimicrobial substances.

  We introduced streptomycin, up to 2,000 units/g and more into the soil (podsol, garden) sometimes to full saturation, and then rinsed the soil with water until no more antibiotic was found in the elutions.

  After this treatment about 1,500 units of streptomycin per gram remained in the soil. Plant seedlings of peas or wheat were planted in such soil and after some time their tissues were analyzed for the presence of the antibiotic. Usually, after 3-4 days and often even after 30-40 hours streptomycin was found in the roots, stems and leaves in amounts up to 10-15 units/g and more. As a rule, soil analyses have shown the absence of the free antibiotic; the latter was probably in the adsorbed state and was actively absorbed by the roots.

  Antibiotic substances entering plant tissues, enhance the bactericidal effect of their sap and thus increase the resistance of the plants to diseases.

  The more abundant the growth of antagonists in the soil, the more antibiotic substances they produce, the more of the latter enters the plants and the stronger the bactericidal effect of the sap becomes.

  The sap of plants which are grown in a sand substrate without microorganisms and without humus is, according to our observations, less bactericidal than the sap of plants which are grown in nonsterile soil rich in humus.

  We enriched the soil artificially with actinomycetes--producers of streptomycin, and we grew in this soil plants--peas and wheat. The sap of such plants was tested for its bactericidal effect on Bac. mycoides and Staph. aureus. Death of the bacterial cells in the sap of the experimental plants followed after 8-12 hours, and in the sap of the control plants which were grown in soil not enriched with actinomycetes, there was only suppression of growth, but death of the bacteria was not observed.

  Plants grown in soil well fertilized with manure or compost had more active sap than the sap of plants taken from unfertilized soil. The sap of plants grown in a greenhouse (corn) was less bactericidal, than the sap of plants, grown in the open ground in the same soil (Krasil'nikov and Korenyako, 1945 a). Eaton and Rigler (1946) observed increased resistance of roots of cotton plant to Phymatotrichum ornnivorum upon treatment of the seeds with carbohydrates. In these cases, according to the author, intense growth of bacterial antagonists in the rhizosphere of the plants was observed.

  Kublanovskaya and Brailova (1954), studied the bactericidal properties of the sap of the cotton plant in relation to the fungus Fusarium vasinfectum and found that its fungitoxicity toward the given fungus was less when the plants grew in soil with antibiotic substances than the fungitoxicity of the control plants growing in soil without antibiotics. The coefficient of multiplication of the fungus in the sap of the control plants was: 13.6 in the germination phase and 11.8 in the phase of cotyledons; in the sap of experimental plants: 7.8 in the phase of germination and 9.8 in the phase of cotyledons. As is seen, the antifungal properties of cotton-plant sap are strengthened at the expense of the antibiotic substances coming from the soil. Accordingly, the plants morbidity due to wilt was less: in the control 96% of the plants were diseased, and among the experimental plants--only 18.4%. Enhancement of antifungal properties of plant sap was also observed by Kublanovskaya in field experiments on plots fertilized with actinomycetal cotton-cake composts.

  Stapp and Spicher (1954, 1955) observed the appearance of protective substance a in the sap of the potato in relation to Bact. phytophthorum during the development of the plant, when the soils were enriched with microbial antagonists.

  The data given show that the plants absorb antibiotic substances from the soil. Antibiotics may be absorbed by the plants not only from solutions of chemically pure substances but also from a complex organic mixture of metabolites, the microbial antagonist.

  Actinomycetes, bacteria and fungi which produce antibiotic substances, grow in the soil in the rhizosphere of plants. They saturate this zone or microfoci in the soil with the products of their metabolism, including antibiotics. The latter enter the plants through the roots and exert their action there. It is self-evident that the concentration of antibiotics in soil, when formed under natural conditions, will be lower than the concentrations created upon artificial introduction. However, under natural conditions, these substances are constantly formed and therefore one would assume that their entrance into plants is not stopped during the whole vegetative period.

  Having entered the plant tissues the antibiotic substances protect them against the penetration of microbial parasites, suppress the growth of those that have already invaded, produce or elevate the toxicity of the plant sap and thus elevate to a larger or smaller extent the immunological properties of the plant.

  In other words, microbial antagonists are factors which increase the resistance and insusceptibility of plants to infections.

  Suggestions on the possible use of antibiotic substances for medical purposes were made by Pasteur, Mechnikov and their contemporaries. Scientists attempted to use bacterial cultures together with their metabolic products for curing the sick. Fehlesein (1883) described a case of curing lupus by introduction of Streptococcus erysipelas into the patient's skin. Colley (1893) used the same organisms for the treatment of a cancer patient. Pavlovskii (1887) introduced a culture of Pneumococcus into the bodies of animals and so prevented them from being infected with anthrax. Bourchard (1889) used the metabolic products of Pseudomonas pyocyane against anthrax. Manasein (1871) and Polotebnev (1872) used the green mold Penicillium in treatment of patients. Many other specialists attempted to use microbial cultures for the purpose of medical treatment. A special branch of medicine, bacteriotherapy even came into existence (cf. Kashkin, 1952; Ermol'eva, 1946; Waksman, 1947; Waksman and Lechevalier, 1953; Kohler, 1955; Korzybski and Kurylowicz, 1955 and others).

  All these investigations had no proper success and recognition and were soon dropped. Only after active substances--penicillin, streptomycin, etc were isolated and chemically purified, did the antibiotic substances produced by microbial antagonists receive general recognition.

  Presently, many antibiotic substances, penicillin, streptomycin, aureomycin, terramycin, subtilin, etc are widely used in therapeutical medicine and veterinary science. This successful use of these preparations in medicine naturally served as a stimulus for work on the use of antagonists and their metabolic products against infections of plants.

  In the foregoing chapter we have shown the beneficent role of microbial antagonists, their inhibition of phytopathogenic organisms in the soil and then protection of plants against fungal and bacterial infections. We noted there that microbial antagonists remove phytopathogenic forms directly from the soil and by virtue of this alone protect plants from diseases.

  However, the antibiotic substances obtained from cultures of antagonists may be used for the removal of phytopathogenic organisms not only from the soil but also within the plant. In other words, these substances should be used as curative remedies.

  What then should be the requirements, in this case, from the antibiotics?

  As in the case of treatment of human beings and animals, antibiotics used in plant growing should: 1) be active against the agent causing the plant disease and have the ability to inactivate toxins; 2) should penetrate easily into the plant tissues; 3) should not be inactivated too rapidly; 4) should exhibit antibacterial activity within the plant tissue; 5) should not be harmful to the plants at concentrations which are toxic to bacteria.

  In addition, the method of use should be technically possible and all the measures should be economically profitable.

  The first point was, in principle, proven experimentally. It was established, that phytopathogenic bacteria and fungi are susceptible to the inhibitory action of antibiotics.

  As was noted above, for each phytopathogenic microbe it is possible to choose its corresponding antagonist and to obtain its antibiotic substances. Among the immense variety of microorganisms existing in nature, producers of antibiotics against bacteria, fungi, actinomycetes, viruses, etc can always be found.

  Concerning the second postulate--the ability of antibiotic substances to penetrate the plants, this question too was answered in the affirmative. In the previous chapter it has been shown that these substances easily enter into the root system and proceed to the aerial parts. Antibiotics can also be introduced through the aerial organs--stems, leaves and seeds.

  The possibility of introducing drugs into plants via the stem, was shown by Shevyrev in 1903. He introduced various antiseptics into fruit trees with the aim of killing parasites. The method developed by him is nowadays often used for extrarhizal nourishment of plants. This method is as follows: a hole is bored in the trunk of the tree and one end of a wick (of gauze or cotton) is placed in the hole; the other end is immersed in a bottle which contains the antibiotic solution; the antibiotic enters the trunk of the tree through the wick and spreads to all parts of the plant.

  In grassy plants antibiotics may be introduced via the stem by simply wetting it or smearing a paste containing the preparation on it. We used the first method. A wetted piece of cotton or gauze was wrapped around the stem of the plant and covered with wax paper, to prevent rapid desiccation.

  We tested the method of introducing antibiotic substances through the trunks of trees on various varieties of fruit-bearing and decorative plants and under different climatic conditions--in Crimea, Caucasus and Moscow (Krasil'nikov and Kuchaeva, 1955). Various antibiotics were introduced into the plants--penicillin, streptomycin, globisporin, aureomycin, terramycin, grisein and other chemically purified preparations. In a few cases we also introduced crude antibiotic substances in the form in which they appear in the culture fluid.

  Experiments have shown that all these antibiotics can enter the plant via the trunk, but in different amounts and at different rates. Penicillin enters at the most rapid rate (see above). Most of the experiments on absorbability were performed with it (Table 126).

  As seen from the table, various woody plants absorb different amounts of penicillin. Some of them, as for example, cherry, sweet cherry, apple, peach and apricot trees absorb antibiotics in large quantities, others like maple, ash tree and lime tree absorb little of it.

  The distribution of the penicillin within the plant also differs. In some plants (cherry, apple, peach, etc) it moves quickly to all parts, into the branches and leaves of the whole crown; in other plants (bird-cherry tree and Halimodendron) it slowly reaches only the lower parts of the branches and leaves and does not reach the upper part of the crown; in still other trees (maple, ash tree and lime tree) it cannot be detected in the leaves at all.

  The intensity of the uptake and the distribution of antibiotic substances in the plant changes noticeably with changes in climatic conditions--temperature, air humidity, soil moisture, etc. The lower the temperature and the higher the humidity of soil and air, the slower is the uptake of antibiotics. For example, in May 1954 in the Nikitskii Garden, when the average temperature of the month was 13.4° C the temperature of the soil 14.2°C and the relative air humidity 92%, peaches and apricots absorbed 45-50 ml penicillin solution each on the first day and during 5 days--100-110 ml. In August, the average daily temperature of the air was 24.3° C the soil temperature was 23° C and relative air humidity was 41%, the same plants took up 200-210 ml on the first day and during 5 days--about 1 liter of penicillin solution (Table 127). In May the absorption of penicillin was 3-5 times less than that in August, although in the spring plant suction is usually higher.

  We obtained similar data in experiments with birch (10 years old) under the Moscow climate. A globisporin solution was administered (activity 5,000 u/ml) to the same plant via the stem on dry and on rainy days. Two repeated experiments were performed: the first in June and the second in August. The amounts of antibiotic absorbed during 5 days were: on dry days in June--1,500 ml, in August--900 ml and on rainy days of the same months the corresponding absorption was 350 and 200 ml, i.e., 4.5 times less.

  In experiments with lemon trees in the orchard of the Institute of Subtropical Cultures (Anaseuli) in the rainy period in September 1952, the antibiotic grisein was absorbed to such a low degree that the work had to be postponed until drier weather set in.

  The rate of distribution of antibiotics within the plant corresponds to the intensity of its absorption. The faster, and the more antibiotic enters, the sooner it is found in the different parts of the plant. In experiments with plants of the Nikitskii Garden we introduced a penicillin solution into trees and the rapidity of its appearance in the leaves 'was measured. Each day after the introduction of the solution 20-30 leaves were removed from the tree and analyzed separately for the presence of the antibiotic in them. Table 128 shows the percentage of leaves in which the antibiotic was detected.

  In August in dry warm weather, the antibiotic is rapidly distributed throughout the whole tree. It can be found everywhere a few hours after its introduction. In May there was cool rainy weather in Crimea. The uptake of the antibiotic and its distribution in the tissues was very weak. Only 2 days after introduction could one detect the antibiotic in the plant's leaves, and then only in some leaves.

  The entrance of the said substances into the plants is connected with the physiological conditions of the latter. The more intense the metabolic processes of the plants, the more vigorous their growth, the faster are the antibiotics absorbed. When the external factors slow down the growth of the plant, the inflow of antibiotics into the roots also slows down. It was noted above, at a lowered air temperature the uptake of active substances is much more sluggish than at a higher temperature in the summer. The same was observed by Stokes (1954) in her work. She determined the rate of uptake of griseofulvin by plants at different temperatures. At 25° C the substance enters the plant 5 times as fast and in larger quantities than at 10° C. She also observed the detrimental effect of excessive humidity on the uptake of the antibiotic. At a 56% relative humidity its concentration in the tissues is 4 times higher than that at a humidity of 91% and a temperature of 25° C.

  Antibiotic substances taken up by the root system, are transported via the xylem to the aerial parts, the leaves. If, however, the antibiotics are introduced through the leaf surface, their transportation is accomplished through the phloem, i.e., as in case of substances synthesized in the leaf.

  Antibiotic substances entering the plant, penetrate inside the cells and cause a certain effect there. In order to follow the penetration of these substances into the cells we used antibiotics which were luminescent in ultraviolet light. Mycetin and certain other substances belong to these antibiotics.

  We allowed the antibiotic solution to pass through the tissues of the plant, we then performed microscopic analyses of microtone slices. Mycetin first enters into the cytoplasm, staining the various granules and rodlike mitochondria and then enters the nucleus, where it reaches higher concentrations than in the cytoplasm

  Some of these substances at certain concentrations inhibit nuclear division upon entering the cells.

  Pramer (1955) followed the penetration of antibiotics--penicillin, streptomycin and chloromycetin into the cells of the algae Nitella Clavata. These algae were immersed in a solution of the antibiotic for some time, were then washed thoroughly, and the cell juice which was squeezed out of the individual cells was collected with a micro-pipette. In this juice the content of the antibiotic which was introduced into the algae was determined.

  It was found that streptomycin and chloromycetin penetrate the membrane, reach the inside of the cells, and spread throughout the protoplast giving the latter bactericidal properties.

  Penicillin, according to the author, is not detected inside the cells. It either does not penetrate them or, if it does, is immediately inactivated.

  Nielsen (1955) found that antibiotics formed by the plankton of water reservoirs suppress the photosynthetic activity of algae of the Chlorella group.

  There are theories which state that upon introduction of various substances into the trunk of a tree, they spread in a sectoral fashion, corresponding to the vascular transport system.

  Taking this in account, we paid special attention to the distribution of antibiotics in the periphery of the bark of woody plants. The administered antibiotic was determined in the leaves and branches located in various parts of the crownaccording to sectors and circular rings-in the lower, middle and upper parts.

  Numerous analyses show quite clearly, that penicillin, streptomycin, grisein and other antibiotics are distributed more or less evenly throughout the crown of the plant. We observed no sectoral distribution of the substances introduced in fruit, ornamental or forest trees.

  Certain antibiotics enter the root system from above and travel downward. According to our observations, grisein possesses this property. When it is introduced into the stem or the trunk of a lemon tree it can be found after a certain time in the lower part of the trunk and in the roots. The tissues contained: in the trunk, at the point where the substance was introduced 120 units/g, near the root 60 units/ g and in the roots-30-50 units/g.

  The method of administering antibiotics through the intact stem by the use of gauze and cotton bandages, was employed by us in experiments with grassy plants and with shrubs; it was also tested with woody plants. Young branches of garden roses, apple trees and pear trees, stems of peas and wheat were wrapped with cotton (or gauze) wetted with a solution of penicillin, streptomycin, grisein or another preparation; after a lapse of some time, the plant tissues were subjected to analysis. As the investigations have shown, these substances penetrated inside the plants, but never accumulated in high concentrations. This method is thus hardly suitable for wide use. However, it may be used for local therapy.

  Introduction of antibiotics through the leaf surface has been performed in experiments with woody and grassy plants. The leaves of the plant were either sprayed with a sprayer or wetted with cotton.

  The spraying of the crown of plants with antibiotic solutions, using a sprayer was employed by us in experiments with fruit trees: peaches, apricots and apple trees and with grassy plants; peas, corn, wheat, etc. Preparations of penicillin, streptomycin and grisein in dilutions of 1:1,000-1:5,000 were used. After some time these antibiotics were determined in the tissues of leaves, branches and stems. Before analysis the severed leaves and branches were thoroughly washed in water.

  Analysis showed the following amounts of antibiotics (in 1 gram tissue):

  The greater the distance from the location of the antibiotic administration, the lower its concentration in the organs of the plant.

  Upon wetting leaf surfaces with pieces of cotton soaked in a solution, even more convincing results were obtained. Two to five hours after the application of the cotton bandages with the antibiotic, the latter could be detected in the leaf tissue which were quite removed from the spot of its application as well as in the petioles of leaves, and even in the tissues of the branches which bear those leaves.

  The American specialists use antibiotics in the form of dust, spraying them on the crown of the plants. The dust particles reaching the surface of the leaves, dissolve and penetrate into the tissues.

  It should be pointed out that in all the experiments with the various methods of introduction of antibiotics, the antibiotics move in the direction of the lower parts as well the upper parts of the plant. Upon introduction of a solution of penicillin or grisein through the trunk of an apricot tree, these substances were detected in the branches, leaves and root tissues as well. The same was observed with peas. A preparation of penicillin introduced through the stem surface, was subsequently found in leaves in the upper parts in a concentration of 5-10 units/g and in the roots, in a concentration of 3-5 units/g (Table 129).

  Mitchell, Zaumeyer, Andersen et al., (1952, 1954) introduced antibiotics, applying them with lanolin paste. The paste with the antibiotics was spread on the stem surface, and after some time the active substance was determined in the tissues of branches and leaves. Brian, Wright et al., (1951) observed the penetration of the antibiotic griseofulvin into plants. Leben, Arny and Keitt (1953) introduced helixin and antimycin into plants, and Hessayon (1951) introduced trichothecin--an antibiotic obtained from the fungus Trichothecium.

  Antibiotics can be used for the sterilization of infected seeds. It is known that in plant seeds there are often phytopathogenic bacteria and fungi which are sources of plant diseases. In order to get rid of these agents, various chemical substances are used--antiseptics. However, the antiseptics which inhibit the growth of microbes also act deleteriously on the seed tissues and decrease their ability to germinate.

  The antibiotics, unlike the antiseptics, act selectively, inhibiting microbial metabolism without causing any harm to the seed embryo. The sterilizing effect of antibiotics was tested by us on cotton seeds. It is sufficient to immerse the seeds for 4-8 hours in an antibiotic solution in order to kill the microbes in the seed tissues (Krasil'nikov, Mirzabekyan and Askarova, 1951; Askarova, 1951: Mirzabekyan, 1952).

  Blanchard and Diller (1951) treated leguminous seeds with aureomycin, allowed them to germinate and then determined the entrance of the aureomycin into the seedlings. The authors noticed a larger accumulation of the antibiotic in the roots than in the upper parts.

  The effectiveness of antibiotics depends on their concentration in the tissues. In turn, the concentration depends on the properties of the plants, especially on the properties of the antibiotic and also on external conditions.

  It was found that. when the solution of antibiotic is concentrated, more of it penetrates the plant (Table 130).

  Very high concentrations of antibiotics (penicillin--5,000 u/ml), grisein--1,000 units/ml) have a toxic effect on plants. They begin to wither and guttation stops. Smaller concentrations are harmless; the plants develop normally.

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