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Part IV, continued:

  Microbial antagonists, an noted above, suppress their competitors with their metabolic products, among which a special place is occupied by the antibiotics. The latter may accumulate not only in artificial nutrient media but also directly in the soil, being concentrated there in smaller or greater amounts.

  It is also known that plants can absorb various organic substances from the medium, including antibiotics, through their roots. If the antibiotic substances enter the plants, it should be assumed that they may produce a certain effect in the tissues, namely, suppress the activity of microbes that have penetrated the cells and increase the toxicity of the cell sap and, consequently, also increase the resistance of the plants to infections. In other words, an assumption is made that the entry of antibiotics is reflected in the immunobiological properties of the plants.

  It should be noted that the problem of immunity in plants, regardless of the numerous studies done on the subject, is still very little known.

  Literature data show that immunity, nonsusceptibility to infections in plants is determined by various, quite complicated internal and external factors.

  One of such internal factors is the toxicity to bacteria of the call sap. It was noted long ago that the sap of plants possesses the ability to suppress growth of bacteria and fungi. Wagner (1915) observed death of bacteria in sap from the tubers and stems of potatoes. Precipitating these juices with ammonium sulfate and washing the precipitate with water, he obtained a substance of a protein nature with strong antibacterial properties. The substance is thermolabile, decomposes quickly in light and under the influence of oxidases and peroxidases. The toxicity of potato juice was observed by Cholodnyi (1939), he found that the toxicity of the juice increases upon sprouting of potato tubers.

  Antimicrobial properties were observed in the juice of onions, corn, tomatoes, cotton, orchids and other plants. In the literature there are descriptions of the inactivation of fungal toxins by the sap of plants resistant to the diseases (Yachovskii, 1935, Vavilov, 1919, Naumov, 1940, Carbone and Arnaudi, 1937). There are indications, that the cell sap from varieties which are resistant to the infection is more toxic than that of susceptible varieties (Kramarenko, 1949; Gorlenko, 1950).

  The antimicrobial properties of vegetative juice are explained by the presence of various substances in the cells.

  An opinion was voiced that plant immunity is caused by the presence of elements of mineral nutrition in the sap. Some of them, potassium copper, cadmium, etc create a nonfavorable milleau in the plant tissues for growth of microbes. According to some authors, they are favorable to the accumulation of organic acid in the cells. Other investigators tried to explain the resistance to infections by the osmotic pressure of the sap, by the presence of alkaloids, enzymes, agglutinins, and lysis, dissolving the microbial cells, etc.

  Israil'skii (1952) expresses the opinion, that the nonsusceptibility of plants to certain infections is caused by bacteriophages which saturate the tissues of the plants.

  Winter and Ruemker (1952) concluded that the nonsusceptibility of plants is caused by the presence of a special "resistance factor" in the roots.

  Many investigators ascribe considerable importance in plant immunity to the redox system (Rubin, 1050, et al.) or to the permeability of the protoplasm of the cells Kuprevich, 194 7, Kokin, 1948; Sukhorukov, 1952), Recently, a connection was noted between plant resistance to infections and the production of pigments of the anthocyanin group.

  On the basis of him extensive studies Tokin (1951) attaches great importance to the phytocides in the immunity of plants. In phytocides are included different chemical antimicrobial substances which are formed by plants. Essential oils and organic acids, aldehydes, alcohols, phenols and also various specific compounds may be included here.

  One would assume that during the course of the history of their development plants acquired various means of protection against infections, mechanical, chemical and biological.

  While ascribing a certain importance to all these, in our opinion, the investigators have not yet touched one of the most important protective factor" the antimicrobial antibiotics, formed in the soil by microbes and absorbed there from the plants.

  It was recently established that in the soil there are antibiotic substances which are formed by the microbial antagonists. The formation of antibiotic substances by microbes in the soil was experimentally proved by many investigators. Gottlieb and Siminoff (1952) have shown the presence in soil of chloromycetin, formed by Actinomyces venezuelae. The substance was isolated from the soil and chemically purified. Owing to favorable conditions of growth of the actinomycetes, 25.0-27.8 mg/kg of chloromycetin accumulate in the soil. The formation of chloromycetin in the soil by A. venezuelae was also noted by Jefferis (1952). The greatest amount is formed upon the addition of peptone or lucerne hay to the soil. In the presence of starch or oat meal the antibiotic is not formed.

  Gregory et al., (1952) observed the formation of antibiotic substances in soil by cultures of bacteria--Bacillus sp. B-6, by actinomycetes--Actinomyces No 67 and by fungi Pencillium patulum. In the presence of appropriate nutrient sources in soil (soy meal) the following amounts of antibiotics were formed:

  Hessayon (1951, 1953) showed the formation of the antibiotic trichothecin by the fungus Trichothecium roseum. Large quantities of the antibiotic were detected in loamy soil, and smaller quantities in sandy soils.

  According to Gottlieb (1952) the antibiotic actidione is formed in the soil in considerable quantities (10 µg/g and more) in the presence of soy meal. Grossbard (1940) has grown Penicillium patulum in the soil in the presence of various sources of nutrition. The formation of patulin took place in the presence of beet cake, glucose, fresh wheat straw, timothy grass and certain other plant residues. According to this author, antibiotics are also formed in the soil by Aspergillus terreus and A. antibioticus in the presence of wheat straw and certain other sources of nutrition.

  In a later work Grossbard (1952) has shown the formation of antibiotics in the soil by the fungi: Aspergillus terreus--70 units; Penicillium sp.--80 units; Aspergillus clavatus--110 units and Penicillium clavatus--100 units in 1 g of soil.

  On the third day of growth of the fungus Aspergillus clavatus, in the presence of brown sugar, Gottlieb and Siminoff (1952) noticed the formation of clavacin in the soil in the amount of 16 µg/g. Taking into account the extent of adsorption and inactivation of the substance by soil particles, the authors determined its total production as not less than 50 µ g/g.

  Stevenson and Lochhead (1954) studied the formation of antibiotic substances by the fungus Penicillium sp. and by the actinomycetes K-1 and V-27 in sterile as well as in nonsterile soil. In sterile soil the fungus forms as much antibiotics as on Chapeks medium (about 20--30 µ g/g and in nonsterile soil 3 times less (7 -l0 µ g/ g). The actinomycetes form up to 16 units/g and more of the active substance. As sources of nutrition both organisms use various plant residues--the green bulk of clover, orchard grass, brome grass and wheat.

  Wright (1955) observed the formation of griseofulvin in the soil by the fungus Penicillium nigricans in the presence of other soil fungi. According to his data, a pure culture of Penicillium nigricans forms the following amounts of the antibiotic (in sterile soils), in podsol at pH 5.3--0.2 µ g on the 7th day and 2.4 µ g on the 14th day, in podsol with Ca(OH)₂--2.4 µ g on the 7th day and 20 µ g on the 14th day; in orchard soil at pH 6.2--0.6 µ g on the 7th day and 10 µ g on the 14th day--in l g of soil. Upon infecting the soil with other fungi the production of griseofulvin either decreases or increases, depending on the species of the fungi. On the 14th day of incubation griseofulvin was detected in the soil in the following quantities:

  In sterile soil which is contaminated with nonsterile soil griseofulvin accumulates much less, namely about 6 µ g/g.

  The author notes that in sterile and nonsterile soils antibiotics are also formed by many other fungi. On the 14th day of incubation in soil infected by fungi he found the following quantities of antibiotics:

  Our studies show that antibiotic substances are formed in the soil by various antagonistic organisms--bacteria, actinomycetes and fungi, if the conditions are suitable.

  In podsol soil in the presence of nutrient sources (soy meal, lucerne or clover straw, sugar or other substances) bacterial antagonists form 40-180 units/g in sterile soil and 10-80 units/g in nonsterile soil. (Table 113). As can be seen from the table, the formation of antibiotic substances is more intense in sterile soils. As an artificial nutrient, here too, the presence of special substances is essential to the formation of antibiotics, and often those are not the same substances as those necessary for the nutrition and growth of the antagonistic bacteria. Of many organic substances tested only some of them were suitable for the synthesis of antibiotics, although growth of the antagonists occurred with any one of the nutrient sources used in the experiments.

   Similar data were also obtained upon testing the actinomycetes-antagonists. The latter, when growing in soils where appropriate organic sources of nutrition are available, form antibiotic substances in detectable amounts (Table 114). We detected these antibiotics directly by the use of microbiological assay methods in the soil, and after extraction with organic solvents. Extraction as a rule, causes lower quantitative results. Where the microbiological assay method indicates 100units/g by extraction one succeeds in isolating not more than 10-30 units/g, i.e., about 10-30% of the total amount present in soil. Depending on the nature of the soil and the properties of the substance itself, the amounts of the extracted substances may vary (Krasil'nikov, 1954c).

  Korenyako, Artamonova and Letunova (1955) studied the formation of antibiotics in soil by actinomycetes belonging to different species. In all more than 100 cultures were studied, 13 of them in great detail. The actinomycetes were grown in soil with the addition of various organic nutrients. All of them, except a very few, formed antibiotics in soil in greater or smaller amounts. The most active ones are presented in Table 115.

  The data given in the table show that microbial antagonists form antibiotic substances with different intensities depending on the type of soil. The largest amount of these substances is formed in podsol soil, less in chernozem and chestnut soils and the smallest amounts in krasnozem.

  The antimicrobial properties of antibiotics express themselves differently in the soil than in artificial media. Not all the antibiotics are active in the soil. Martin and Gottlieb (1955) have shown, that circulin, neomycin and biomycin do not suppress the growth of the sporiferous bacterium Bac. subtilis in the soil even at very high concentrations (500 µ g/g), whereas on laboratory nutrient media they inhibit its growth at 0. 1 µ g/g. At the same time subtilin noticeably inhibits the growth of Bac. cereus in these very same soils. In these author's experiments antinomycin was the most active. Five µ g/ g actinomycin in soil was a sufficient amount for the suppression of Bac. subtilis growth.

  The effectiveness of antibiotics in the soil is determined not only by their properties but also by their concentration. In turn the latter depends on the rate at which these substances are formed and enter, the soil on the one hand, and by the rate of their inactivation on the other hand. As is known, the majority of antibiotics disappear from the soil. Some are inactivated by the soil solution or destroyed by microbes, others are washed out by water and a considerable portion is adsorbed by soil particles.

  in our experiments (Krasil'nikov, 1954c) we followed the degree of activity of antibiotic preparations, introduced artificially into various soils under different conditions. The rate of inactivation of antibiotics, the extent of their being washed out by water and their adsorption by the soil were studied. The average indexes chernozem soil are presented in Table 116.

  Up to 2,000 units/g of chemically pure preparations were introduced into the soil. Preparation 1609 was introduced at a concentration of 10,000 units/g.

  As is seen from these data, streptomycin, globisporin and terramycin are inactivated in chernozem to more or less the same extent (about 25%), penicillin, to a lesser extent and preparation No 1609 is completely inactivated. In other soils the inactivation of these antibiotics presents a different picture (Table 117). The least inactivation of these antibiotics is observed in podsol soils and in krasnozem, and the greatest in chernozem.

  In order to determine what part of antibiotics are adsorbed by the soils, we washed the latter with water. Upon introduction of antibiotics into podsol soil in the amount of 2,000 units/g the following portions could be washed out with water: penicillin, 1,650 units/g; streptomycin, 40 units/g; globisporin, 120 units/g; terramycin, 350 units/g, and the antibiotic No 1609--none.

  Penicillin more than other antibiotics can also be washed out of other soils; e, g., krasnozem, 1, 800 units/ g and from serozem, 1, 500 units/ g.

  By comparing the numerical indexes of inactivation of the antibiotics and their proneness to be washed out by water, the amount of antibiotics in the adsorbed state can be determined and therefore also the adsorbing capacity of the soil. According to our data in podsol soil the adsorption picture was as follows: globisporin--1,800 units/g; streptomycin--1,880 u/g, terramycin--1,350 u/g, penicillin--280 u/g per gram. In serozem the figures were 1,670, 1,600, 1,200 and 380 units/g respectively. Streptomycin is adsorbed by chernozem at the rate of 1, 120 units /g and by krasnozem--1,650 units/g; globisporin--1,080 and 1,540 units/g and terramycin--900 and 1,620 units/g respectively.

  As seen from this data the amounts of antibiotics detected are considerably smaller than those introduced. In order to create antibacterial activity in 1 g of serozem soil for example, it is necessary to use a minimum of 350 units streptomycin; 100 units globisporin; 600 units terramycin; 130 units penicillin and more than 10,000 units preparation No 1609. Upon introduction into the soil of smaller antibiotic doses than those indicated, the antimicrobial properties of the soil will not be detected either by a qualitative test or by extraction with various solvents (water, alcohol, ether, acetone, etc). All these data show that to the numerical indexes obtained upon quantitative determination of antibiotics formed in the soil by microbial antagonists, one should add the amounts of antibiotics adsorbed and inactivated. If 20 units/g of antibiotic substance were found in the soil, the actual amount produced by the antagonists would be considerably higher.

  The rate of destruction of antibiotics in the soil varies. Some of them are inactivated within a few hours and others may be preserved for a few days or even weeks, depending on the nature of the substance and the properties of the substrate.

  Antibiotics with basic properties such as streptomycin are very quickly inactivated in the soil, Neutral compounds (chloromycetin) are inactivated slowly, and substances of the acid type occupy an intermediate position.

  Adsorption and inactivation of antibiotic substances depend to a considerable degree on soil acidity. Aureomycin and terramycin saturate neutral loamy soils at a concentration of 30,000 µ g/g, while for the saturation of acidic loam 60,000) µ g/g and more are required, i.e., twice as much.

  At pH 3.2 soil rich in humus adsorbs 4,000 µ g/g of antibiotics and at pH 5.6-7.6 only 400 µ g/g, i.e., ten times less.

  Consequently, the antimicrobial action of antibiotics will differ in these soils. In order to inhibit growth of Bac. polymyxa in soil at pH 5.6 a concentration of 5,00 µ g/g of terramycin is required, while at pH 6.2, 200 µ g/g suffice (Gottlieb and Siminoff, 1952; Martin and Gottlieb, 1952).

  The stability of antibiotics in the soil also varies with the acidity of the latter.

  According to Gregory et al., (1952), the antibiotic actidione is preserved in alkaline soil at pH 7.8 for 8 days and in an acidic soil at pH 5.2--for more than 14 days. Clavacin to completely inactivated in the first day in alkaline soil and in acidic soil it is preserved for 3-4 days. Ninety per cent of fradicin is preserved in alkaline soil for 14 days, in an acid soil it is adsorbed and completely inactivated an the first day.

  Chloromycetin is less strongly inactivated in the soil than are streptomycin and terramycin. When chloromycetin is introduced into sterile soil, it remains for more than 14 days without change. In nonsterile soil, with a large number of various microorganisms, the preparation is gradually inactivated; after 3 days only 70% of it is left, after 7 days--about 30% and after 2 weeks only 20% are recovered.

  Pramer and Starkey (1052) have found, that streptomycin introduced into the soil at a concentration of 1,000 µ g/g is preserved in sterile soil for over 3 weeks and in nonsterile soil for 2 weeks. About 50% of it is destroyed within the first week. In the presence of glucose the antibiotic is preserved for a longer period of time.

  Jefferis (1952) tested many antibiotic substances. He introduced them into various soils and observed the rate of their destruction. The following data have been obtained (Table 118).

  An may be seen, in orchard soil there is a faster inactivation of antibiotics. According to the author, certain substances are destroyed faster in sterile soil than in nonsterile soil, which is puzzling and disagrees with the observations of other investigators.

  According to our data (Krasil'nikov, 1954c), the preservation time of antibiotics varies greatly and depends first of all, upon the properties of the substances, and secondly, on the type of soil and external conditions (temperature, humidity, acidity, etc), The same antibiotic is inactivated and parishes at different rates in different soils. For instance, globisporin is preserved for 7 days in podsol, but for only 2 days in krasnozem. Aureomycin is active 10 days in podsol but not more than 3 days in krasnozem (Table 119).

  Antibiotics are most rapidly inactivated in krasnozem and podsol soils.

  Preparation No 1609 disappeared immediately in nonsterile soils; other preparations could still be detected after a few days. In sterile soils antibiotics are preserved for considerably longer periods of time than in nonsterile soils. As can be seen from Table 119, the majority of active substances disappear in the first 2-3 days in nonsterile soils, while in sterile soils they are preserved for 2 -3 months.

  Similar data are obtained upon introduction of crude antibiotics in the soil. Korenyako, Artamonova and Letunova (1955) introduced active actinomycetal substances in the form of culture liquids into podsol, chernozem, krasnozem and serozem soils. About 700 crude preparations were examined, 12 of them, in greater detail. The results are given in Table 120.

  The soil solution exerts a slightly inactivating effect. We tested a solution obtained by using a strong press from incubated samples of podsol, serozem and chernozem soils, with 4 antibiotics: penicillin, globisporin, preparation No 15 (grisein) and preparation No 1609. The soil solution was added in various amounts to the antibiotic solution of a known titer and after a certain period (1-5 hours or more) the activity of the preparations were examined. The results were not always clear-cut, however, reliable data were obtained in experiments with grisein and, preparation 1609 and in some cases also with penicillin. The solution from incubated podsol soil inactivated antibiotics to a lesser degree than solutions obtained from serozem and chernozem. One ml of solution extracted from podsol neutralized 20-30 units of preparation No 1609, 5-7 units of grisein and 2-5 of penicillin. Soil solution from incubated serozem inactivated 50-60 units of preparation No 1609, 7-10 units of grisein and 10-12 units of penicillin. Solution of incubated chernozem inactivated 80, 15 and 25 units, respectively. In addition it also inactivated approximately 5-7 units of globisporin.

  The inactivating force of soil solution is closely related to the species makeup and metabolism of the microorganisms. If one incubates soil in the presence of' small amounts of organic substance and in addition inoculates the soil with certain bacterial species, the solution thus obtained would inactivate considerably more antibiotics. We incubated podsol soil with various bacterial species (sporeformers and nonsporeformers) with an admixture of various sources of organic nutrition (soy meal, clover, hay, corn extract, peptone), The greatest effect was obtained in an experiment with a nonsporeforming bacillus (strain No 6) which was incubated in soil with soy meal. The solution obtained thereof (1 ml) inactivated 100 units of preparation No 1609 and up to 50 units of penicillin, but did not inactivate grisein or globisporin. The latter was inactivated by solution from soil incubated with bacterium No 15 in the presence of clover hay.

  The inactivating action of the soil solution was observed by Waksman and Woodruff (1942), They examined the effect of actinomycin in pure solution and with an admixture of a humus extract. A culture of Bac. mycoides was killed in the former case by 1 µ g and in the latter case, by 10 µ g or more of the substance. A similar effect was observed by Skinner (1956) while studying the antibiotic obtained by him from Actinomyces albido-flavus.

  Inactivation of antibiotics in soil is probably mainly by products of microbial metabolism. It has been shown, that with increase of the latter, destruction of antibiotics, is enhanced. In the above-mentioned experiments, in soil incubated together with soy meal, the growth of bacteria was very intense and their composition differed from that in soil with clover or peptone.

  Winter and Willeke (1951 a, b) introduced penicillin into composted soil rich in humus and into loamy soil poor in organic substances. In humus soil, where there was abundant growth of microbes, the antibiotic disappeared after 2-3 hours. In soil poor in humus the same antibiotic was preserved for 12 hours. If the soil microflora is removed by sterilization, penicillin in such soil is preserved for more than 3 days, while in nonsterile soil it disappears on the second day.

  We have demonstrated the inactivating role of the soil microflora in experiments with pure cultures of bacteria and actinomycetes. It was found, that the antibiotics, penicillin, mycetin and streptomycin are inactivated in different degrees by metabolic products of various species of bacteria and actinomycetes. Some species or strains of bacteria inactivate streptomycin more strongly, while others inactivate penicillin and mycetin more strongly. There are cultures the metabolic products of which enhance the activity of antibiotics (Krasil'nikov and Nikitina, 1951).

  The data given here change our concepts on the preservation of antibiotics in soil. Antibiotics are not always fully inactivated in the soil. Depending on the soil-climatic conditions and also an the chemical properties of the antibiotics themselves, they may be preserved and accumulate in the soil.

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