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Part IV, continued: 

  These data show how large and versatile is the microflora which produces biotic substances. With the aid of these substances, the microorganisms growing in the soil activate the growth, nourishment, and many other vital processes of both higher and lower organisms.

  In soils, as well as in other natural substrate, there are microorganism-inhibitors. which, in course of their metabolic activity, suppress the growth and development of higher and lower plants. They form special substances which are toxic to plant tissues and organs. Toxins, or phytotoxins formed by phytopathogenic fungi and bacteria, were studied long ago by numerous investigators (see Kuprevich, 1947; Sukhorukov, 1952; Bilai, 1953, Gorlenko, 1953; Goiman, 1954). However, the question of whether these organisms produce toxic substances directly in soil remained unsolved in the literature. It is known that many species of fungi and bacteria form toxins which act on animal organisms. Growing on food products, fodder, and on various plant residues, they excrete toxic substances. Upon feeding these products to animals, one often observes a strong case of poisoning (Pidoplichko, 1953).

  Investigations show that microbial inhibitors may poison plants with their toxins under conditions of their natural growth in soil, if favorable conditions for such growth are formed. They suppress germination of seeds, the growth of sprouts and plant growth in general and decrease the total crop. Consequently, when there is a massive growth of these organisms, they may become an important factor in determining the fertility of soil and the crop yield of plants.

  The suppressing action of microbial inhibitors is also manifested in the growth of lower plants--fungi, bacteria, algae and others. In such cases, the microbes are called antagonists.

  Microbial inhibitors are found among various groups of lower forms: bacteria, fungi, and actinomycetes. Greig-Smith (1911) established the fact that toxic substances formed by certain bacteria, suppress the growth of plants. Ressel (1933) ascribed great importance to Protozoa, which consume microbial cells. Hutchinson and Thaysen (1918), Lewis (1920), and Laudenberger (1952) noticed in certain nonsporiferous bacteria of the genus Pseudomonas the ability to synthesize potent toxic substances. Johnson and Murwin (1931), and later Braun (1950), discovered this ability in Pseudomonas tabaci, the causative agent of tobacco disease. The ability to form toxic substances was also found in other bacterial groups.

  Among the group of nonsporiferous bacterial inhibitors, representatives of the genera Bacterium and Pseudomonas, are comparatively often encountered and lose often those of the genus Rhizobium. They are often found in the rhizosphere of vegetative plants. We studied more than 300 cultures of these organisms isolated at different times from different soils, from the chestnut soils of the Trans-Volga region, the serozem soils of Central Asia, and the podsol soils of the Moscow and other regions.

  Of this number of cultures which were studied, about 100 suppress to a greater or smaller degree the growth of plants and the germination of seeds, Strongly expressed herbicidal properties were possessed by certain strains of Ps. flourescens, Ps. pyocyanea and Bacterium sp. They completely or almost completely inhibited the germination of needs of clover, vetch, and wheat (Figure 86). The seeds merely sprouted and died, or did not show any signs of germination.

Figure 86. Suppression of the germination process of clover seeds by nonsporiferous bacterial inhibitors, Posudomonas sp.

a--control; b--in the presence of bacterial inhibitors.

  The toxic properties of many sporiferous bacteria are sharply expressed, We studied more than 350 cultures, isolated from various soils of the Soviet Union. The bacteria were grown in liquid nutrient media. The seeds were treated by soaking them for several hours in the culture fluid. Seeds of plants treated with culture fluid were germinated on cotton or on paper which had been wetted with water.

  The toxic or herbicidal effect of the bacterial fluid revealed itself in the suppression of growth and the lowering of the percentage of germinating seeds, Analyses have shown that approximately 20-30 per cent of the cultures investigated possessed inhibitory proportion. Among the bacteria isolated from turfy podsol soils, the number of inhibitors was large (about 34-45 per cent).

  The nature and strength of their effect vary in different cultures. Some organisms completely or almost completely inhibit the germination of seeds (Figure 87), others are less inhibitory, and still others do not show any inhibitory effect whatsoever.

Figure 87. Effect of sporiferous bacterial inhibitors on the germination of plant seeds. The seeds were soaked in the bacteria-culture fluid and germinated on cotton or on paper wetted with water:

A--effect of Bac. mesentericus (strain 50) on germination of wheat seeds: 1--control, seeds soaked in water; 2--seeds soaked in culture fluid; B--effect of Bac. mesentericus (strain 67) on the germination of seeds of peas: 3--seeds soaked in culture fluid; 4--control, seeds soaked in water.

  The species of the inhibitors studied by us mainly belonged to Bac. mesentericus and Bac. subtilis.

  The capacity to suppress the germination of seeds and the growth of seedlings is revealed in various degrees among strains belonging to the same species. Among cultures of the bacterium Bac. messentericus, we found more than 180 strains isolated from various soils, including 100 strains from the podsol soil of the Chashnikovo Experimental Station. Among these were some very strong inhibitors, while others did not inhibit plants growth at all. Some of them inhibited the germination of wheat seeds, others those of peas, vetch or clover, while some of thorn inhibited the germination of wheat, peas, vetch and clover seeds. In Table 95 data are presented from an experiment with vetch and clover.

  Among sporiferous and nonsporiferous bacteria, there are sometimes strains possessing an organotropic or selective herbicidal action. They either suppress the growth of only this root system or of only the aerial parts. We found cultures which completely inhibited the growth of the roots of vetch and wheat. The seeds sprouted without the formation of roots, while the latter were very much reduced (Figure 88). The aerial part developed more or less normally as long as the seeds contained nutrient substances.

Figure 88. Suppression of growth of wheat roots by bacterial inhibitors:

a--experiment; b--control.

  Some bacterial strains in our collection (three nonsporiferous and four sporiferous strains) inhibited the growth of the aerial parts, but did not affect the root system. The needs germinated a root, while the aerial part was strongly reduced (Figure 89).

Figure 89. Inhibition of growth parts of vetch by cultures of bacterial inhibitors:

a--experimental, b--control plant.

  Certain strains of bacteria suppress the sporulation process of lower organisms, the formation of zygotes in phycomycetes and the formation of spores in yeasts (Krasil'nikov, 1947 a). It is possible that there are microbes which inhibit the fruiting process of higher plants as well.

  In our collection of actinomycetes there are strains which cause chlorosis of higher plants by the action of their metabolic products. Chlorosis appeared in corn and wheat after treating the seeds before sowing with a culture fluid of certain species of actinomycetes and, even more markedly, with purified preparations of antibiotics. If the seeds of these plants are kept in a solution of an antibiotic for two, to four hours before sowing, the seedlings are completely colorless, without the slightest sign of the formation of chlorophyll. The growth of much plants is suppressed and, soon ceases altogether. In some cases the plants, recover, become green, and continue to grow more or less normally,

  If the seeds are treated with weaker solutions of the antibiotic, one obtains seedlings which are slightly green and somewhat etiolated. Strongly etiolated plants are obtained upon the treatment of seeds with streptomycin. Soaking seeds for two hours, in a solution of one microgram per ml causes the complete etiolation of the seedlings. The latter do not become green for a period of 15-30 days and finally die. A suppression of chlorophyll synthesis is caused by aureomycin, terramycin and other antibiotics.

  We obtained the etiolation of duckweed by growing it in a nutrient solution to which an antibiotic had been added. Depending on the concentration of the antibiotic, the growth of the plants was inhibited to a greater or smaller degree. The extent of the appearance of the green color also varies, from slight chlorosis to full colorlessness.

  Certain toxins of microbial origin cause the phenomenon of chlorosis in grapevines, According to our observations, this phenomenon may be due to fungi of the genus Fusarium. We found certain strains, the toxins of which caused the etiolation of shoots, of cuttings, and grape stock, when treated before planting in the soil. The plants that grew from them had light green leaves with a yellowish hue, their development was slow, and other deviations were observed which are characteristic of chlorosis of grape vines (Krasil'nikov and Kublitskaya, 1956).

  This picture of the etiolation of cuttings was observed by us after the treatment of the vine with antibiotics of actinomycete origin. Certain strains of gray and pigmented actinomycetes synthesized substances which inhibit the formation of chlorophyll in the leaves of grapevines. Cuttings, when immersed with their basal ends in the crude fluid culture and subsequently planted in the soil developed and showed obvious signs of etiolation.

  The inhibition by antibiotics of the formation of chlorophyll in plants has been mentioned by certain other investigators. Provasoli, Huntner, and Schatz (1948) obtained colorless cultures of Euglena sp. under the influence of streptomycin. The antibiotic was added to the nutrient solution in small quantities; under its influence the chloroplast of the cells was destroyed, as a result of which completely etiolated forms of organisms were obtained.

  The phenomenon of chlorosis as an effect of streptomycin was observed in cereals (wheat, corn, etc) by Von Euler (1947) and Hagborn (1956). They wetted the seeds of plants in the antibiotic solution and planted them in the soil. The seedlings were devoid of green color.

  Berezova and Sudakova found that the death of the growing tip of flax is not connected with boron starvation, but is the result of poisoning by toxins formed by bacteria.

  Kugushova has shown that on the roots of lucerne, bacteria may grow which, by their excretions, cause the failing off of the buttons (according to Berezova, 1953 a).

  Inhibitors, suppressing the growth of plants and the germination of seeds, are encountered in great numbers among actinomycetes. In this group of microorganisms, cultures with strong herbicidal properties are most often found among the orange A. aurantiacus, among the gray A. griseus, and among other species and groups (Table 96).

  Experiments with seedlings have shown a more or less similar picture. Some cultures of actinomycetes strongly suppress growth, while others only slightly or not at all (Table 97).

  Toxic substances of actinomycetes and other microorganisms exert a suppressing effect on single isolated organs or parts of plants; on leaves, cuttings, etc. If one immerses the cuttings, or cuts off the leaves, then, after a certain period of time, they wither and die. By the speed of the withering and death of these parts one may judge the strength of the action of the toxic substances.

  In our experiments we used cuttings of various plants, of beans, peas and corn, and branches of lemon, apple, pear, and apricot trees, etc.

  If one puts on the surface of an uncut leaf a piece of cotton which has been wetted with toxin, after a few hours spots appear of a necrotic nature. The stronger the poison, the more sharply the necrotic spots on the leaf are expressed. This method was used by us in testing the toxic substances formed by microorganisms.

  Among the inhibiting factors of great importance are the phages: bacteriophages and actinophages. The studies of Rautenshtein (1955), Khavina (1954), and certain others show that these agents are widely distributed in soils, where they are detected in considerable numbers. There is reason to believe that they suppress and lyse cells of bacteria or actinomycetes as readily as under conditions of pure cultures.

  For example. root-nodule bacteria become inactive when phages multiply abundantly in the soil. Under conditions of the experiment, they multiply to a considerable extent. We have counted tens and even hundreds of thousands in one gram of soil. According to Demolon and Dunez (1934), phages of root-nodule bacteria of clover and lucerne, under certain conditions, saturate the soil to such an extent that the soil becomes much less fertile for these plants, root-nodule bacteria do not develop in it, and there is only a slight or no formation of nodules on their roots, and when they do develop they have an abnormal appearance. The authors are of the opinion that the observed clover-lucerne soil exhaustion is caused by the accumulation of phages. According to certain data, phages penetrate the plant, and, by interfering with plant metabolism, lower crop production (Vandecaveye and others, 1940).

  There is data in the literature on the formation of toxic substances by fungi. Leng (1949) has shown the poisoning effect of the Penicillium fungi on the seedlings and of cereals. The most active inhibitors in these experiments were P. notatum, and P. oxalicum. Monnaci and Torini (1932) and Diachum (1934) note the formation of toxins by fungi, which act on cereals under conditions of their growth in soil.

  Producers of toxic substances are known among various groups of soil microflora. An important place is occupied by representatives of the genus Fusarium. The substances formed by them were obtained in a chemically pure form having a known structure; for example, lateritin, C₆H₄₆O₇N₂ ; avenacein, C2₅H₄₄O₇N₂; fructigenin, C₂₆H₄₄O₇N₂; sambucynin, C₂₄H₄₂O₇N₂, and enniatins, lycomarasmin, yavanicin, etc.

  These substances act differently on plants and animals. Some of them are specific (Goiman, 1954).

  Fusaria are very widespread in nature. The probably play an important role in the toxicoses of soils. Their inhibitory effect on the growth of plants was observed by many authors (Rehm, 1953; Laundoldt, 1952; Sukhorukov, 1952). The significance of these fungi for the fertility of soils is not only determined by their ability to synthesize toxins and excrete them into the soil but also by their phytopathogenic properties.

  Bilai (1955) described in his monograph many strains of the genus Fusarium which have a deleterious effect on the germination of seeds and on the growth of seedlings of rye, oats, and barley. The products of their metabolism, obtained in the form of filtrates, were tested under various conditions. The results of the author's experiments are given in Table 98.

Table 98
Effect of filtrates of Fusarium cultures on the germination of plant seeds
(in length of plant parts in cm)

Fungal culture	Rye, rootlets	Rye, sprouts	Barley, rootlets	Barley, sprouts
Control	21.5	4.25	29.8	3.6
Fus. poal., strain 2	3.8	1.9	--	--
Fus. poal., strain 5	8.3	2.6	16.0	3.6
Fus. poal., strain 9	11.7	2.5	11.8	2.3
Fus. poal., strain 41	2.4	1.6	--	--
Fus. poal., strain 45	15.0	5.4	8.4	1.2
Fus. sporitrichioides, strain 28	6.1	1.4	18.4	2.1
Fus. sporitrichioides, strain 30	11.3	3.2	6.0	1.5
Fus. sporitrichioides, strain 51	15.3	6.3	11.2	1.5

  As can be seen from the table, the filtrates of some strains affect the seedlings of rye, while others act predominantly on the growth of barley. Certain strains suppress the growth of rye and wheat to the same extent as that of barley or oats.

  Klechetov (192 6) in studying the phenomenon of the flax exhaustion of soils found the growth of the fungi Fusarium, Thielaviopsis basicola, Cladosporium herbarum, Alternaria, and Macrosporium in these soils; these fungi, according to the author, form toxic substances and are the reason for the death of the sown flax.

  A considerable role in the exhaustion of soils and in the lowering of plant yields is attributed in the literature to the fungi of the genus Fusarium. Kvashina (1938), Kurtesova (1940), and Ioffe (1950).

  Kublitskaya (1955) studied the degree of the distribution of fungi of the genus Fusarium in the soils of Central Asia (Uzbek SSR) under grapes. She isolated 52 cultures and many of them proved to be toxic for grapevines, causing poisoning and death to the cuttings and stock under the conditions of growth in soil. Certain strains caused chlorosis under experimental conditions.

  Strongly expressed herbicidal properties are exhibited by fungi of the genus Pythium. According to Likais (1952), Pythium debaryanum forms toxins in the soil which inhibit the root systems of plants.

  Mirchink (1950) studied a large collection of fungi isolated from turfy podsol soils of the Moscow district and found among them many toxigenic forms. The most toxic and the most widespread fungi in these soils are representatives of the genus Penicillium and, secondly, Fusarium and Trichoderma. Fungi of the genus Trichoderma (T. lignorum) and certain representatives of the genus Fusarium strongly suppress the germination of wheat seeds, as a result of which the number of germinating seeds decreases by 68 per cent and more. The length of sprouts in the presence of the metabolic products of Trichoderma is 3.5 cm; in the presence of the fungus Fusarium, 4.0 cm; and in the control, 4.6 cm. The Penicillia inhibitors are often found in the turfy podsol soil in a great number of species. Some of them are very toxic for wheat, which can be seen in Table 99 and in the photograph (Figure 90).

Figure 90. Effect of the culture fluid of the fungus Penicillium nigricans on the germination of wheat seeds:

a--control; b--treated seeds.

  Active toxin producers in soil are fungi of the genera Trichoderma, Trichothecium, Botrytis, and others. From cultures of Helminthosporium (H. victoriae), the toxin victorin was isolated, which inhibits the growth of roots and seedlings of oats at a dilution of 1:1,000,000. This substance is formed by the fungus directly in the soil (Weeler Luke, 1954; Tyler, 1948). Toxic substances harmful to plants were found among the representatives of Verticillium. The most well studied among them is V. alboatrum. Its toxic substance was found by Bewley (1922), It causes the withering of tomatoes, cotton, tobacco, and other plants. Green (1954) discovered two substances in this fungus-- a protein and a polysaccharide. The former is excreted into the medium and the latter enters the tissues of the plants, The poisoning effect of this fungus was also noted by Sukhorukov (1952) and others,

  Among members of the genus Trichothecium were found the toxic substances trichothecin and others, which inhibit plants and certain microbes, Similar substances were found in Deuterophoma tracheiphilus, causing "malsecco" in citrus plants, They were also encountered in many other fungi (Hossayon, 1953; Freeman and Morrison, 1949, Gelman 1954).

  It is obvious that the importance of microbial inhibitors in soil toxicosis will be mainly determined by the degree of their growth and activity.

  The distribution of microbial inhibitors and their accumulation in the soil has been but occasionally studied; as were microbial activators. Monnaci and Torni (1932) found about 60 per cent of the soil fungi isolated and investigated by them, to be inhibitors.

  According to our data, there are a great number of inhibitors among the fungi, bacteria, and actinomycetes in soils. Out of 1,500 cultures of actinomycetes, more than 200 inhibited, to a larger or smaller degree, the germination of beet or wheat seeds and 16 strains completely suppressed their germination; 21 cultures strongly suppressed and 58 weakly suppressed the growth of clover and lucerne, The total number of inhibitors among actinomycetes is comparatively small, on the average 5-15 per cent,

  One finds inhibitors among sporiferous bacteria considerably more often, Out of 560 strains studied, belonging mainly to three or four species, Bac. mesentericus, Bac. subtilis, Bac. cereus, and Bac. brevis. 178 strongly suppressed the germination of clover seeds, more than 200 cultures suppressed to some degree the germination of peas. According to our data, there are about 40 per cent inhibitors among the sporiferous bacteria of Bac. mesentericus and Bac. subtilis isolated from the turfy podsol soils.

  Inhibitors among nonsporiferous bacteria are encountered much less frequently than among sporiferous bacteria, According to our calculations, their number can be expressed in a tenth part of one per cent. Some species of the genus Bacterium and Pseudomonas possess, however, strongly expressed toxic properties in relation to plants and microorganisms.

  It should be noted that certain microorganisms among bacteria and fungi react to toxic substances in the same way as do higher plants, which enables us to use them as test organisms in the screening for and the study of phytotoxins. Microbial tests have a number of advantages, With them one can more quickly determine and solve a number of problems related to the toxicosis of the soil and the poisoning of plants, In mass studies we often use both tests; the microbiological and the plant test.

  We carried out the quantitative evaluation of microbial inhibitors in different soils, but went into greater detail in the turfy podsol soils of the Moscow Oblast', the Kola Peninsula, and in other regions of the USSR. Virgin and cultivated soils, forest and swampy soils, meadows, ate were investigated,

  We counted from 5,000 to 450,000 inhibitors in one gram of soil depending on the properties of the latter (Table 100). In slightly cultivated soils, the absolute number of inhibitors is smaller, but its percentage may be higher than in wellcultivated soils.

Table 100
Number of inhibitors in podsol soils of Moscow area
(Number of cells in 1 g of soil)

Soil	Bacteria inhibiting azotobacter	Actino- mycetes inhibiting azotobacter	Fungi inhibiting azotobacter	Bacteria inhibiting beet seedlings	Actino- mycetes inhibiting beet seedlings	Fungi inhibiting beet seedlings
Dolgoprodnoe
Virgin soil	15,000	23,000	1,300	8,000	3,000	500
Plowed fields	45,000	17,000	2,000	15,000	7,000	1,000
Agricultural Academy Timiryazev
Forest	40,000	80,000	17,000	25,000	10,000	4,000
Virgin soil	10,000	32,000	1,500	10,000	3,000	500
Plowed fields	120,000	150,000	2,300	50,000	35,000	3,000
Chashnikovo
Forest	120,000	82,000	12,000	20,000	12,000	7,000
Virgin soil	40,000	16,000	1,600	10,000	5,000	1,000
Plowed fields	450,000	160,000	1,400	150,000	60,000	500

  Inhibitors which suppress the growth of Azotobacter in podsol soils are much more numerous than microbes which suppress plant growth. Mirchink (1958) studied the fungal flora of soils of the experimental station Chashnikovo (Moscow Oblast') and found that 11-38% were inhibitors which suppress plant growth. They were distributed in the following manner: in forest soil--13%, in glades--11%, in cultivated soils, 15-38% of the total microflora, detected by by existing methods (Table 101).

Table 101
Number of fungi in podsol soils
(thousands in 1 g of soil)

Soils	Total	Inhibitors, %
Control soils without fertilizers	60	32
Fertilized with mineral nitrogen	138	38
Calcium-containing fertilizers + manure	36	24
Calcium-containing fertilizers + manure + P.K.	18	15

  As can be seen from the data given, the greatest number of inhibitors was found in soils cultivated to a limited extent. Mineral fertilizers do not diminish but, on the contrary, they noticeably increase the content of inhibitors.

  It was experimentally established that microbial inhibitors form toxic substances directly in the soil in which they grow.

  If these organisms are introduced into nontoxic or inactivated soil and the soil is incubated under certain conditions of humidity and temperature, then after a certain time it will become toxic for these or other plants or for certain species of microorganisms, depending on the peculiarities of the inhibitor.

  Rybalkina (1938 a) observed the appearance of toxicosis in flax-exhausted soil upon growth of the fungus (Fusarium lini).

  Mirchink (1956) incubated soil (podsol) with fungi-inhibitors and she observed the appearance of toxicosis. In soils in which the fungus Penicillium cyclopium grew abundantly if artificially introduced, seeds of wheat did not germinate at all or germinated in small numbers (Figure 91). Other species of fungi isolated from podsol soils also poisoned the soil but to a lesser degree. On such soils germinating wheat seedlings constituted 15-60% of the number of seedlings in normal control soil.

Figure 91. Poisoning of soil by cultures of fungi upon artificial infection. Germination of wheat seeds:

a--in control (noninfected) soil; b--in soil in which Penicillium nigricans grew; c--in soil infected with Penicillium cyclopium.

  Clover-exhausted soil inactivated by heating regains its toxicity by growing the appropriate microbial inhibitors in it. In such soil with regenerated toxicity, clover and root nodule bacteria grew much more poorly than in normal soil. There was either no nodule formation on the root of clover or it was considerably suppressed (Table 102). Root-nodule bacteria in such soil became avirulent and lost the capacity to penetrate the roots and form nodules in them. Cultures of some fungi of the genera Penicillium, Fusarium, Trichoderma and some sporeforming bacteria, when growing abundantly in inactivated forest or field soil restored the soil's original toxicity in relation to wheat and Azotobacter (Table 103).

Table 102
Growth of clover on soil with restored toxicity

Expermiental conditions	Number of sproutings	Number of sprouts on the 30th day	Number of nodules per plant (average)
Inactivated soil not infected with inhibitors (control)	46	46	23
Inactivated soil, infected with inhibitors:
Ps. pyocyanea	32	26	0.05
Ps. tumefaciens	39	22	0.0
Fusarium sp.	41	31	0.5
Mixture of all bacteria	30	19	0.0

Note: Each vessel contained 50 seeds. Inoculation was performed with active cultures of Rhizobium trifolii.

Table 103
Accumulation of toxic substances in podsol soil an a result of growth of inhibitors

Name of inhibitor	Length of wheat rootlets, cm	Length of wheat sprouts, cm	Survival of Azotobacter cells, hours
Control: inactivated soil (not infected)	12	8	240
Infected with:
Bacillus strain 12	3	5	6
Bacillus strain 23	6	2	12
Bacillus strain 8	4	4	16

  Toxins produced by inhibitors may, under certain conditions, accumulate in considerable quantities and endow the soil with toxic properties. The extent of accumulation of toxic substances depends upon the intensity of their formation by microorganisms, the rate of destruction and leaching, and also upon the degree of adsorption.

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