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Part III, continued:

  The capacity of microorganisms to synthesize biotic substances has been known for a long time,

  Wildiers (1901) showed the presence of activating substances in yeast cultures, These substances were called by the author bios substances, Fifteen to twenty years later the attention of various specialists was drawn to these substances, They were found in various organic substrates, in plant tissues and in cultures of many microbes.

  Investigations showed that biotic substances are synthesized by various microorganisms--bacteria, fungi, yeasts, actinomycetes and others (Meisel, 1950, Ierusaimskii, 1940 Kudryashov, 1948; Bukin, 1940, Stephenson. 1951, Schopfer, 1943).

  The organisms are divided according to their capacity to synthesize biotic substances into auxoautotrophs and auxoheterotrophs. The former synthesize all the compounds necessary for their growth and can therefore grow on synthetic, vitaminless media; the latter do not synthesize, or more precisely, they do not synthesize all the necessary biotic substances and therefore they cannot grow on vitaminless media,

  The capacity to synthesize various growth factors is present in many if not in all species of soil bacteria.

  Chemosynthetic bacteria are the most active in synthesizing biotic substances, They can grow in purely mineral, completely vitaminless media, being capable of synthesizing organic substances from atmospheric CO₂. For example, thiamine, riboflavin, pantothenic acid, nicotinic acid, vitamin Be and some other compounds were found in the culture of Thiobact. thioxidans (O'Kane, 1943). These bacteria and other similar chemosynthetic bacteria--the nitrifters and hydrogen-and methane-oxidizing bacteria, synthesize their full quota of biotic substances. Without this ability they could not have developed in mineral media,

  Bacteria incapable of CO₂ assimilation, but growing well in synthetic vitaminless media with organic carbon sources, are also able to synthesize biotic substances, To such bacteria belong the bulk of the soil microflora Azotobacter, root-nodule bacteria, representatives of the genus Peoudomonas, Bacterium, oligonitrophils, mycobacteria, and others.

  Boysen-Jonsen (1931) found that hetroauxin is synthesized by 16 bacterial species among which were Ps. radiobacter, Bact. denitrificans, Bac. mycoides, Bac. subtilis, Bacterium sp., and others. Raznitsyna (1938) employing the coleoptile method has detected the formation of this compound by various representatives of bacteria and mycobacteria. She divided microorganisms into three groups, according to their ability to synthesize auxins: a) organisms which do not synthesize them (or synthesize them in small quantities) Mycobacterium rubrum, Az. vinelandii, Bac. mycoides, and others, b) bacteria of medium activity --Az. agile, Az. chroococcum strains 31, 35, Bact. coli, Myob. luteum, Ps. flourescens, strains F. 24 and others, c) bacteria synthesizing large amounts of auxins--Bact. proteus, Ps. fluorescens strain 21, Az. chroococcum strain 54, Mycob, album, and others.

  Roberts I, and Roberts E. (1939) studied the capacity of bacteria, fungi and actinomycetes to synthesize heteroauxins. This compound was synthesized by 99 cultures out of a total 150 studied, According to the authors, the most active producers of heteroauxins were bacteria and actinomycetes. Heteroauxin was found in different species of nonsporeforming bacteria--Az. chroococcum, Pseudomonas, Bacterium, Vibrio, Mycobacterium, and many other bacteria,

  Beside auxins, a number of other biotic compounds such as thiamine, riboflavin, para-aminobonzoic acid, nicotinic acid, pantothenic acid, folio acid, vitamin C, vitamin K, vitamin B₃, vitamin B₁₂, inositol, biotin, provitamin D₂, alpha- and ßcarotenes, factor R, factor Z, and others can be detected in bacterial cultures (Detinova, 1937, Leo and Burris, 1943, Jones and Grooves, 1943; Burton and Lochhead, 1951, Lochhead, 1952; Lochhead and Burton, 1955), The ability of the root-nodule bacteria to synthesize thiamine, riboflavin, pantothenic acid, vitamin B₁₂ and others was discovered by Burton and Lochhead (1951), Went and Wilson (1938, 1939). These and other vitamins were found in cultures of Az. chroococcum, in various species and strains of the genus Pseudomonas and in mycobacteria. In recent years vitamin B₁₂ was found in many bacteria and especially in actinomycetes, Some of these organisms such as Mycob. propionicum, A. rimosus, A. aureofaciens, and others, are employed in industry for the production of this vitamin. According to our observations, 90-95% of the actinomycetes isolated from soil synthesiss vitamin B₁₂ (Krasil'nikov, 1954 c), According to Darken, 64-66% of soil actinomycetes synthesize this vitamin, (Darken, 1953).

  Yamagutschi and Usami (1930) found vitamin B₂ in 15 bacterial cultures (Bac. subtilis, Bac. Mesentericus, Bac. mycoides, Bact. prodigiosum, various micrococci, and others).

  Landy, Larkum and Oswald (1943) found paraminobenzoic acid in cultures of 35 species of bacteria and actinomycetes. Among these were: Bact. proteus , Ps. pyogenes, Bac. aerogenes, Bac. subtilis, Bac. megatherium, Mycob. diptheriae, Mycob. stereosis, and others.

  Herrick and Alexopoulus (1943) found thiamine in cultures of 22 species of bacteria and actinomycetes.

  According to Schmidt and Starkey (1951), 99 species of bacteria and actinomycetes, out of a total of 150 species studied, synthesized heteroauxin. Twenty-two bacterial cultures out of a total of 75 cultures studied, synthesized vitamins on synthetic media.

  We have studied 192 bacterial cultures isolated from different soils of the USSR. These cultures were tested for their capacity to synthesize vitamin B₁ and heteroauxin. The results are given in Table 42. It can be seen from the table that more than 50% of the bacteria studied synthesize vitamin B₁ and almost 40% synthesize heteroauxin.

  Shavlovskii (1954, 1955) studied cultures of Ps. fluorescens, Ps. aurantiaca, Bact. herbicola and Ps. radiobacter from the rhizosphere of plants. These bacteria were grown in synthetic media of a defined composition and after 8 days incubation the vitamin contents of the bacterial cells and of the culture filtrate were determined. The results are given in Table 43,

  Many fungi produce biotic substances. Thus, for example, thiamine was detected in cultures of Aspergillus niger, A. oryzae, Penicillium glaucum, Mucor mucedo, Mucor racemosus in some species of Phytophithora, Rhizopus, Fusarium and others; and also in cultures of yeasts such as Torula utilis, Sacchar. Cereviseae, Sacchar. logos, Endomyces vernalis, Willia anomala (Bilai, 1955; Goinman, 1954; Bukin, 1940, and others),

  Fungi have been found in the soil which synthesize vitamin B₂. Some of them synthesize this vitamin in such quantities that they are used in industry. These are Candida (Oidium), Guilliermondella, Eremothecium ashbyi (Dikanskaya, 1951). Biotin, pantothenic acid, nicotinic acid, paraaminobenzoic acid, vitamins C and K, and many others have been found in many fungi (Meisel, 1950).

  Rogosa (1943) studied 114 various yeast cultures such as Torula sphaerica (26 strains) T. cremoris (20 strains), Sacch. fragilis, Monilia pseudotropialis, Mycotorula lactis, Sacch. anamensis, Torulaopsis kefyr, Torula lactosa, Zygosaccharomyces lactis and others. In all the cultures they found vitamin B₂ in amounts ranging from 0.6 to 0.11 µ g per ml of synthetic medium.

  Biotic substances were also found in the mycorhiza fungi. According to Schaffstein (1938), mycorhiza fungi of orchids synthesize growth factors required for the normal growth of the host plant.

  Biotic compounds are also synthesized by algae in the soil. It was mentioned above that these organisms are widely distributed in soil. Frequently algae grow on the earth's surface, forming a blue-green coating, visible to the naked eye.

  It is known that algae secrete various organic substances--metabolic products (Goryunova, 1950). Biotic substances are among these products.

  Ondratschek (1940) found ascorbic acid, (vitamin C) in the secretions of such algae as Hormidium borlowi, H. flaccidum, H. nitens and H. stoechidium. The green alga Chlorella synthesizes heteroauxin (Lilly and Leonian, 1941).

  The majority of soil microorganisms are partially auxoautotrophs, i. e., they require only some biotic substances. For example, Clostridium butyricum requires only biotin and synthesizes its other requisites. Some bacterial species require only thiamine or pantothenic acid. According to Burcholder, McWeigh and Mayer (1944) of 163 yeast cultures 87% required biotin, 35% thiamine and pantothenic acid and 12 % required only inositol.

  There are many bacteria which can synthesize only a fraction of a vitamin's molecule. For example, some organisms synthesize only part of the thiamine molecule, either thiazole or pyrimidine components of vitamin B₁. Consequently, the former would require pyrimidine and the latter--thiazole.

  There are many soil microbes which require ß-alanine, desthiobiotin. pimelic acid and some other compound s-- components of this or other molecule of a biotic substance (Meisel, 1950; lerumalimskii, 1949; Stephenson, 1951, and others).

  Thompson (1942) has shown that bacteria synthesize vitamins in amounts exceeding those required for their metabolism. The excess of vitamins is released into the environment. The enrichment of the substrate with vitamins takes place, not only at the expense of decomposing cells, but also through the secretion of vitamins by living organisms. The possibility in not excluded that some biotic substances are waste products of growing cells.

  According to Thompson, about 50% of the synthesized thiamine remains in the cells of bacteria in a bound state. This fraction finds its way into the soil only after the death and decomposition of the cells.

  An idea on the quantitative aspect of the synthesis of biotic substances by microorganisms can be obtained from the data in Table 44. These data have been compiled from various sources.

  According to West and Wilson (1938, 1939) there are 19.6 µ g thiamine and 0.37 µ g riboflavin per 1 g of dry weight of the root-nodule bacteria of clover, grown on a synthetic medium. Clostridium (Clostr. butyricum) synthesizes 0.9 µ g/g riboflavin and Micro. ochraceus, Micr. citreus, Ps. pyocyanea, about 10-15 µ g.

  Yamagutschi and Usami (1939) found about 1.5 µ g thiamine per g of dryweight of cells in cultures of Ps. fluorescens, Ps. alba, and Bact. prodigiosum. In cultures of Bact. proteus they found 9-14 µ g thiamine per 1 g dry weight of cells.

  Considerable quantities of biotic substances are synthesized by many mycobacterial species. For example, Mycob. smegmatis synthesizes about 135 µ g Oof vitamin B₂ per ml of synthetic medium and 36 µ g per g of the dry weight of cells (Mayer and Rodbart, 1946). Forty to eighty µ g per ml of medium. of the active substance mycobactin was found in cultures of Mycob. phlei (Francis at al., 1953).

  Ostrowsky and others (1954) studied vitamins in various representative microorganisms. Their results are given in Table 45.

  The above-given quantitative data are not strictly constant. They may vary, depending on the growth phase and culture conditions of the individual species of the bacteria, fungi or actinomycetes. In some cases old cells contain less vitamins than young cells, whereas in some species the reverse picture is observed: the old cells contain more vitamins than the young cells. For example, some cultures of Ps. radiobacter contain more vitamin B₁ after 8 days growth than after 2 days growth.

  In some media the microbes synthesize many growth factors, in others only a few or none.

  The intensity of the formation of vitamins by soil organisms is greatly influenced by the symbiotic microbes. Some of them suppress vitamin synthesis and other stimulate this process. According to Smalii (1954), Azotobacter (Az. chroococcum) in pure culture synthesize 173 µ g of heteroauxin (per cell mass in 1 Petri dish, on Ashby agar) and in the presence of the following microorganisms synthesizes this substance in the noted amounts:

Bact. mycoides, 220 µ g
Bact. denitrificans, 196 µ g
Ps. radiobacter, 243 µ g
Torula rosea, 234 µ g
Act. Coelicolor, 188 µ g
Penicill. Nigricans, 149 µ g

  The capacity to synthesize auxins or vitamins does not characterize a species Different strains of one and the same species differ markedly from each other. For example, of more than 100 strains of Az. chroococcum which we isolated in different soils and places in the USSR, some of them synthesized large quantities of heteroauxins and others synthesized only small quantities, or none at all. Formation of heteroauxin by the different representatives of these cultures in shown in the coleoptile photograms (Figure 63).

Figure 63. The formation of heteroauxin by different cultures of Azotobacter chroococcum. The coleoptile curvature after immersion in the culture, expressed in degrees:

a) museum strain 54, angle of deviation--32°; b) strain isolated from garden soil in Moscow vicinity, angle of deviation10°; c) strain isolated from the soils of Kara Kum, angle of deviation-8°; d) strain isolated from cultivated podsol soil (Experimental Station Chashnikovo, Moscow Oblast, angle of deviation--0°; 1--control coleoptile; 2--experiment immersed in the bacterial culture.

  Similar data were obtained when studying other bacterial species and not only for heteroauxin but also for biotin, thiamine, riboflavin, and other biotic substances.

  Although there is no strict species specificity as far as the synthesis of biotic substances is concerned, nevertheless mass analysis does show group differences in this respect. More strains of Azotobacter synthesize vitamins and auxins than bacteria of the genus Bacterium.

  Only a few species of root-nodule bacteria are capable of synthesizing heteroauxin and even these are weak forms. We have investigated 12 species of root-nodule bacteria of clover, lucerne, kidney beans, vetch, Lathyrus vermus, lungwort, peas, Onobrychis, soya, lupine, acacia and astragalus. All these species either did not synthesize heteroauxin at all, or synthesized it in small amounts only (Figure 64).

Figure 64, The formation of heteroauxins by various species of root-nodule bacteria. The magnitude of the curvature on immersion in cultures of:

a) red clover, the angle of curvature-6°; b) soya, angle of curvature- 6°; c) broad beans, angle of curvature- 3°; d) peas, angle of curvature-4°; e) vetch, 4°• angle; f) sweet clover, 2° angle; g) beans, 2° angle, h) proactinomycetes from the nodules of alder tree, 3° angle; 1--control coleoptile; 2--experiment immersed in bacteria.

 

  Out of 60 strains of root-nodule bacteria of lucerne, only 9 strains synthesized this compound in amounts able to give a barely perceptible coleoptile curvature. Fifteen strains of Rhizobium trifolii, 8 strains of Rhizobium leguminosarum and 15 strains of Rh. phaseoli were examined. In all cases the picture was the same.

  We have not detected any synthesis of heteroauxins by proactinomycetes which form nodules on the roots of alder tree; actinomycetes and proactinomycetes of the soil do synthesize heteroauxin to a greater or lesser extent.

  According to Starkey (1944), the nicotinic-acid content of plant residues ranges from 2.4 to 85 µ g per gram of dry weight, in the majority of cases it is lower than 30 µ g/g. The same substance in microbial cells amounts to 150-1,920 µ g/g, i. e., approximately 25-60 times more.

  It should be noted that the studies of biotic substances were carried out on relatively few species of soil microorganisms. The choice of organisms was taken at random, and the studies were confined to a few vitamins only, in the majority of cases to thiamine and riboflavin.

  It should be assumed that in reality many and possibly all soil microorganisms synthesize these or other biotic substances which play an essential role in the life and metabolism of lower and higher organisms.

  It is obvious that under natural conditions (life in the soil) the microbial metabolism and the synthesis of biotic substances would differ from that under laboratory conditions (on artificial nutrient media).

  Schmidt and Starkey (1951) have shown that if plant residues which do not contain vitamins are introduced into soil which also does not contain vitamins, the latter appear and accumulate in greater or lesser amounts due to the decomposition of the residues by microbes. The increase in the riboflavin content of the soil is concomitant with the intensification of microbial metabolism (Figure 65). The more plant residues introduced into the soil, the more intense the microbial growth and the formation of riboflavin (Table 46).

Figure 65. The formation of riboflavin in the soil as a product of the metabolism of microorganisms, the activity of which is determined by the evolution of CO₂ in mg per 100 g of soil:

1--riboflavin, in µ g/100 g; 2--CO₂ in mg/100 g.

  Similar results are obtained if glucose or saccharose are introduced into the soil instead of straw; the bacteria inoculated into the soil lacking the vitamins begin to grow at the expense of the sugars; and riboflavin, biotin, heteroauxins, etc accumulate in the soil.

  According to Meisel's calculations (1950), about 400 g of vitamin B₁, 300 g of vitamin B₆ and 1 kg of nicotinic acid are synthesized by microbes in the surface layer of one hectare of the fertile soils of the southern regions, during one season (9 months).

  Biotic substances are preserved in the soil for varying periods of time. A pure preparation of a vitamin introduced into the soil can be detected for several days. According to Schmidt and Starkey (1951), riboflavin and pantothenic acid persist in the soil from 3 to 20 days or longer (Table 47).

  Riboflavin persists in soil longer than pantothenic acid. Both compounds last longer in sterile than in contaminated soil, since biotic substances, like all other compounds, are subjected to microbial decomposition.

  If the microbial metabolism is artificially arrested, vitamins introduced into contaminated soil persist for the same periods as in sterile soil. It was found that biotic compounds (vitamins and heteroauxin) persist in samples of dry soil taken from cultivated and fertilized fields, from 3 to 4 months to 4 years depending on the kind of soil and its properties and also on the properties of the vitamins themselves (Stewart and Anderson, 1942).

  Vitamins and other biotic substances entering the soil by one or another route are decomposed and synthesized de novo by microorganisms, Some vitamins disappear others appear. There is a continuous turnover of these substances in the soil. Biotic substances can be found in the soil during the entire vegetative period an long as the microbes live, reproduce and exhibit metabolic activity. The amount of the biotic substances is determined by the rate of their synthesis and introduction into the soil and also by the rate of their destruction, or their stability, 

  It was noted above that green plants synthesize for themselves the necessary biotic substances or phytohormones. Under conditions favorable for their growth this synthesis meets all their requirements for normal growth. In certain, not infrequent, circumstances, apparently under some unfavorable conditions, the plant synthesizes inadequate amounts of these substances. Then specific avitaminoses develop which are expressed to a greater or lesser degree in the form of certain physiological disturbances and diseases.

  Different plants react variously to the addition to the substrate of growth factors and vitamins. Some respond by enhanced growth or by changes in the course of biochemical processes, others react weakly and still others do not react at all. This permits us to assume that the first produce only minimal amounts of the active substances which are insufficient for their normal metabolism, the second synthesize them quite actively but in amounts still insufficient to satisfy all their needs, and the third synthesize them in adequate quantities.

  Investigations show that even the last group of plants by no means always synthesize adequate amounts of biotic substances. The vitamin content of plants varies within a wide range, depending on external conditions of growth. It varies according to the soil and climate conditions (Murry, 1948; Rakitin, 1953). Fertilizers have a great effect on the quantity of vitamins present in plants.

  In all cases of avitaminosis the vitamins from the substrate are absorbed by the plant. Even under normal conditions of growth, plants utilize ready-made biotic substances, if available.

  The utilization of vitamins, auxins and other compounds from the soil has been confirmed in many experiments. Many plants and biotic substances were studied under laboratory and field conditions, in sterile and nonsterile experiments.

  The plants' requirements for vitamins and auxins has been thoroughly studied in experiments with isolated organs and tissues, and especially with isolated roots.

  It is known that excised roots of many plants will not grow in synthetic media in the absence of biotic substances and a carbon source. If a root 2-3 mm long is excised from a plant which grew under sterile conditions and placed in a synthetic artificial medium (Bonner's medium or other) it will grow in length to reach considerable dimensions and will form lateral roots, etc, only if the necessary biotic substances are present in the medium. In the absence of the latter, or if their concentration is insufficient. the roots will not grow at all or the growth will be weak.

  Investigations show that roots of different plants demand different growth factors. For example, roots of flax require vitamin B₁, roots of peas, horse-radish, lucerne, clover and cotton require vitamins B₁ and B₆; roots of tomatoes. thorn apple, and sunflower require vitamins B₁, B₆ and pantothenic acid (Bonner et al., 1937; Robbins and Bartley, 1922-1938; Robbins and Schmidt, 1939, 1945). Excised roots of many plants, growing on synthetic media, synthesize all the required growth factors. Some of them synthesize them in amounts sufficient for their normal growth, others form too little. The first grow well in artificial media, the latter require the addition of the missing factors (Bonner, 1942). Bonner and Bonner (1948) give the following data on the vitamin requirements of isolated roots (Table 48).

Table 48
Vitamin requirements of isolated roots of various plants
(according to Bonner T. and Bonner H., 1948)

Plants	Vitamin B1 requirement	Nicotinic acid requirment	Vitamin B6 requirement
Linum usitatissimum Boenn	stimulates	-	-
Raphanus sativus L.	+	+	-
Medicago sativa L.	+	+	-
Trifolium repens L.	stimulates	+	-
Gossypium hirsutum L.	+	+	-
Crepis rubra L.	+	+	-
Cosmos sulfureus	+	+	-
Pisum sativum Gov.	+	+	-
Daucus carota L.	+	-	+
Lycopersicum esculentum Mill	+	-	+
Lycopersicum esculentum pimpinellifolium	+	stimulates	+
Dun	+	stimulates	+
Helianthus annuus L.	+	stimulates	+
Acacia melanoxylon R. Br.	+	stimulates	+
Datura stramonium L.	+	+	+

  The following data show the effect of vitamins, on the growth of isolated roots. The roots of flax in the presence of vitamin B₁ elongated by 185 mm. and in its absence by 31 mm in one week. The roots of flax are calculated to synthesize vitamin B₁ at a rate of 0.02 µ g per week. Their vitamin B₁ requirement for normal growth is 2 µ g, i.e., 100 times more than they synthesize.

  The roots of white clover grow well in a medium containing vitamins B₁ and PP*. *[ The correct designation of this vitamin is unclear.] They increase in length with each successive transfer into a fresh nutrient medium. In the first 5 weeks the roots elongate by 84 mm, the increment in the next 5 weeks amounts to 109 mm, in the third five-week period the increment amounts to 129 mm, in the fourth five-week period--136 mm, and in the following 5 weeks 151 mm. The increment becomes uniform upon subsequent transfer amounting to about 22 mm per week.

  The roots of sunflower in the absence of the vitamin complex or in the presence of only one of the vitamins PP or B₆ cease to grow after 7 consecutive resowings. Roots which were supplied with all three vitamins, PP, B₁ and B₆ grew well for along period of time allowing for many transfers into fresh media. In the first five weeks the increment was 74 mm, in the next 5 weeks it amounted to 96 mm, in a further 5 weeks--120 mm, and in the following fortnight--150 mm.

  The roots of plants belonging to diverse varieties of one and the same species react differently to vitamins. For example, one variety of tomatoes requires vitamin B₆ and does not respond to vitamin PP and on the contrary another variety requires vitamin PP and does not react to vitamin B₆ (Bonner and Bonner, 1948).

  Ovcharov (1955) introduced vitamin PP into a medium in which he grew cotton plants whose leaves were cut off. He observed enhanced formation of new roots on the old roots.

  Went, Bonner and Warner (1938) had shown that thiamine stimulates the growth of roots of peas, lemons and camellia. The results were more markedly pronounced when a mixture of thiamine and heteroauxin was employed. Positive results were also obtained in these cases with a mixture of vitamin B₁ and indoleacetic acid (Grebenskii and Kaplan, 1948) and also with vitamin K, biotin and pantothenic acid with biotin (Scheurmann, 1952).

  Psarev and Veselovskaya (1947) noted the stimulating effect of thiamine on the formation and growth of wheat roots.

  In some cases roots of certain plants required only parts of the vitamin molecule, for example only thiazole or pyrimidine (components of vitamin B₁).

  Some roots require unknown biotic compounds and cannot, therefore, be grown in vitro.

  Robbins (1951) in his review, brings a list of plant species the roots of which can grow on nutrient media. There are 22 such species. Roots of 27 species could not be grown in isolation despite the addition of various vitamins, auxins, amino acids and other biotic substances.

  It should be noted that even the roots which can grow in vitro do not grow in the same manner as when attached to the plant. They grow in length and branch, but do not get thicker or if they do thicken, then only very slightly. The activity of the cambium is completely or almost completely suppressed. Consequently, no entirely adequate medium has as yet been found for isolated roots.

  The requirement for biotic substances is well pronounced in seedlings. The need embryos of some plants develop better and quicker in the presence of certain vitamins added to the substrate. For example, the growth of pea seedlings separated from the cotyledons considerably increases in the presence of thiamine and biotin (Kögl and Haagen-Smit, 1936). Pantothenic and ascorbic acids also act favorably on pea embryos (Bonner T. and Bonner H. , 1948).

  Plant embryos do not synthesize biotic substances, they utilize the food reserves present in the seeds. Even the green sprouts of many plants in the early growth period synthesize vitamins weakly or not at all (Bonner et al., 1939). Ripe embryos of thorn apple are easily grown on artificial media without vitamins while the nonripe embryos require vitamins PP, B₁, B₆, C and others.

  Pantothenic acid also has a favorable effect on lucerne sprouts. Treating pea seeds with vitamin C enhances their growth by 213% as compared to the controls. Sprouts of meadow grass react positively to the addition of vitamins B₁, PP, H and pantothenic acid to the medium. The grape seeds germinate quicker in the presence of an 0.01 % solution of vitamin PP and, moreover, the formation of roots and the growth of aerial parts is more intense (Flerov and Kovalenko, 1947).

  The presowing treatment of cotton seeds with vitamins B₁ and PP considerably enhances their germination and the subsequent growth of the sprouts, Seventy-five per cent of the seeds germinated after treatment with the vitamin as compared to 45 % in the control. The length of the sprouts in the former case was on the average 1.35 cm and 1.65 in the latter Zakharyants, Gorbacheva and Zglinskaya, 1950).

  An increase in the growth and subsequent yield of bean seeds after treatment with vitamins B₁ and PP was observed. The height of the plants (from the treated seeds) was greater by 18%, the increment of the vegetative mass was greater by 39%, and the yield of seeds was 28% higher then that of the control plants (Dagis, 1954).

  Bonner et al. reached the conclusion that the lower the vitamin content of plant leaves, the stronger they react to the addition of these substances. According to them, peas and tomatoes, contain 13-18 µ g of vitamin B₁ per kg of dry leaves and do not react to its addition. Cabbage, cosmos, Japanese camellia and others contain small amounts of vitamins in their leaves and react positively to the addition of these substances. However, this does not hold for all plants. There are species or even varieties of one and the same species which contain a small amount of vitamins in their leaves and react less to their addition than plants with higher vitamin content.

  The addition of vitamins has a favorable effect even on mature plants. Tung trees after the addition of 0.5 mg of vitamin B₁ grew in 70 days twice as much as the controls. The application of low concentrations of vitamin B₁ to poppies increased the weight of their bolls as well as the crop in general. Application of vitamin B₁ together with water had a favorable effect on the growth of spinach. The weight increment during the 63 days of the experiment exceeded many times that of the control plants (Table 49).

  Denisov introduced vitamin B₂ into a substrate where egg plants were grown and obtained a marked increase in yields. After 77 days growth the control plants had stems 7.1 cm long, the weight of the tops was 48 5 g and the weight of the roots 12.5 g. The corresponding figures for plants grown in the presence of the vitamin B₂ were 12.2 cm 121 g and 22.9 g (Ovcharov, 1955).

  The growth of vine grafts, soya and other plants in increased under the influence of vitamin PP. Lemon seedlings react markedly to the addition of this vitamin (Table 50).

Table 50
The growth rate of lemon seedlings under the influence of vitamin PP
(according to Kocherzhenko and Snegirev, 1946)

Treatment	Average height of plants on 15/ VIII	Average height of plants on 15/ IX	Average height of plants on 15/ X	Average height of plants on 15/ XI
Control	27.2	29.2	35.5	38.3
Vitamin PP	23.8	34.7	44.5	47.7

  Analogous data were obtained by Matveev and Ovcharov (1940) in their experiments with a Bukhara almond. The plants were sprayed with an aqueous solution of vitamin PP and adenine. Earlier opening of buds and more rapid development of' leaves were observed. The number of leaves was 4 times greater than in the control plants.

  Vitamins play a considerable role in the development of orchids. These plants, as already mentioned, grow badly, or do not grow at all without the micorhizal fungi. It was found that the seeds of orchids contain only small amounts of vitamin PP, which are not sufficient for normal germination. This shortage is remedied thanks to the mycorhizal fungi. Treating the seeds with vitamin PP secures their normal germination in the absence of the fungi. The dry weight increment was 3 times higher than that in control plants. It was shown that orchids of the group Vanda grow well in the presence of substances obtained from the mycorhizal fungi. These substances resemble, in their action, bios II (according to Kelly, 1952). According to Noggle and Wynd (1943), some orchids grow well in the presence of nicotinic acid. Henrikson (1951) noted the positive effect of thiamine, vitamin B₆ and nicotinic acid on the germination and subsequent growth of Thunia marschaliana Rchb. f. (Table 51).

  Rakitin and Ovcharov (1948) employed vitamin PP and adenine for increasing the growth of cotton plants in their early growth stages. Thereby, not only growth, but also fruiting was increased. The number of bolls was increased, and the cotton yield (raw material) was considerably higher than that of the controls. Similar data were obtained by Zakhar'yants, Gorbacheva and Zglinskaya (1950). They sprayed cotton plants with solutions of vitamin PP and thiamine, The cotton crop increased by 34.1% as compared to the control.

  The fat-soluble vitamins, A, E, K, in contrast to vitamins of the B-group suppress the growth and diminish the crop of plants. Carotene suppresses the growth of safranin which is itself rich in carotene. Vitamin K suppresses the growth of fungi, some bacteria and the roots of higher plants. Vitamin PP antagonizes the action of vitamin K. Vitamin E, according to Schopfer (1950), arrests the growth of certain plants. The height of plants in the control was 35.75 cm and in the presence of vitamin E--6.14 cm; the number of flowers in the former was 80.4 and in the latter, 10.

  Ovcharov (1955) immersed the seeds of plants in a solution of thia ine and yeast extract, Such procedure markedly stimulated the growth and increased the crops, the seeds too were larger.

  Söding, Bömke and Funke (1949) obtained 30% higher yields of carrots after treating their seeds with nicotinic acid, vitamin B₁, vitamin C and other substances.

  Experiments with vitamins under sterile conditions are worthy of mention. McBorney, Bollen and Williams (1935) tested the action of pantothenic acid on the growth of lucerne under sterile conditions in sand cultures, in a medium which did not contain nitrogen. Pantothenic acid was added in high concentrations. Plants under these conditions grew in the presence of pantothenic acid ( in high concentrations of pantothenic acid) much better and the yield was higher.

  Magrau and Mariatt, (1950) showed that a number of vitamins, such as thiamine, nicotinic acid, biotin and pantothenic acid had a positive effect on the growth of Poa annua L. under sterile conditions. Swaby (1942) tested the effect of certain organic substances including some containing vitamins, on the growth of cereal and leguminous plants, in the presence and absence of microorganisms. The experiments showed that in the presence of microorganisms organic substances rich in vitamins have a favorable effect on the growth of plants.

  Shavlovskii (1954) tested the effect of pantothenic acid, vitamin B₁, nicotinic acid and vitamin B₆ on the growth of lucerne. The latter was grown on agar medium under sterile conditions for 30 days. The results are given in Table 52.

  Analogous experiments were carried out by Shavlovskii with buckwheat. The plants were grown in sand wetted with the nutrient solution of Hellrigel. containing 1 µ g of the vitamin per ml. Other containers were supplemented with yeast extract and vitaminless casein hydrolysate. In one series of experiments the bacterial culture of Ps. aurantiaca--vitamin producers were introduced. Plants were grown for 2 days and then analyzed. The results are given in Table 53.

  It can be seen from the given data that the substances tested, markedly increase the increment of the roots and aerial parts of the plants.

  The biological role of vitamins has been little studied, but, according to the available data, it is important. It is well known that many of them are components of various enzymatic systems. The so-called coenzymes which enter into chemical interaction with the substrate include many vitamins, It has been found experimentally that vitamin B₁ in a compound together with phosphoric acid is the coenzyme of carboxylase-cocarboxylase.

  Carboxylase is an enzyme participating in transformations of carbohydrates. It is widely distributed in plants, animals and microbes. Without it the various transformations of carbohydrate compounds, including pyruvic acid, are not feasible. The latter is the key intermediate in the metabolism of living cells, which links the metabolism of carbohydrates, proteins and fats.

  In the absence or shortage of vitamin B₁ the synthesis of cocarboxylase is slowed down or arrested and, consequently, carbohydrate metabolism is slowed The latter is frequently arrested at the stage of pyruvic said, which leads to the accumulation of pyruvic acid in the cell and the complete cessation of metabolism.

  Vitamin B₁ participates not only in decarboxylation of pyruvic acid but also in the reverse reaction--the fixation of CO₂ in pyruvic acid. The role of vitamins in the fixation of CO₂, as the investigations of recent years have shown, is very great.

  Vitamins also play a considerable part in the formation and transformation of proteins. It has been shown that vitamins B₂, B₆, B₁₂, PP and H participate in the formation of amino acids and their transaminations. The shortage of vitamin B₆ leads to a decrease in the formation of amino acids from organic acids and ammonia. Vitamin B_e takes part in the formation of amino acids from organic acids and ammonia. Transamination, i.e., transfer of an amino group (NH₂) from one acid to another, takes place in the presence of vitamin B₆.

  In fat synthesis from sugars, vitamins B₁, B₂, PP and pantothenic acid participate, and the transformation proteins into fats also requires vitamin B₆.

  Vitamins play an immense role in respiration. It was shown that enzymes participating in respiration consist of proteins and a coenzyme. The latter consists of vitamin B₂ and phosphoric acid. Vitamin B₂ in enzymatic systems plays a role in oxidation-reduction processes. Folio acid is of great importance in respiration. The germination of seeds and the respiration of sprouts increases under the action of this acid (Stephenson, 1951, Schopfer, 1943, Zeding, 1955).

  Growth stimulators--auxins and heteroauxins--have a positive effect on the colloidal and chemical properties of protoplasm. According to some authors, they participate in the general metabolism of the cell as separate components. By increasing the metabolism they influence the growth of the cells of the aerial parts and especially of the roots. Under the influence of heteroauxin the influx of plastic substances increases which leads to the formation of now roots in greater quantities. During the rooting of grafts, hydrolysis of starch and fats increases in the cells of the latter. The activity of peroxidase is also increased and the tissues are better hydrated (Maksimov, 1940, Turetskaya, 1955, Zeding, 1955, and others). The action of these substances does not affect the turgor of cells only, as previously assumed, but affects the general metabolism of the plant. In this respect they resemble other biotic substances. Data exist which show that heteroauxin stimulates the formation of auxins, (Zeding, 1955).

  Kuhn (1941) has shown that carotene and carotenoids have a great effect on the formation of sexual cells and on conjugation of a Chlamydomonas alga. According to him, there exist carotenoids with specific properties of male and female hormones. He found a carotenoid--safranol--with properties of a male hormone and a carotenoid--picrocrocin--with the properties of a female hormone.

  Vitamins have a favorable effect on the fertilization of plants. It was found that the sexual organs are rich in vitamins especially the pollen. For example, the pollen of the pea tree contains 2,300 mg of carotene and that of sunflower, 1,460 mg per kg. Pollen of some plants do not contain large amounts of carotene. Vitamins decompose under the action of light and pollen decolorizes and loses its activity. Processing of such pollens with carotene increases its capacity to germinate. Thus, according to Lebedev (1952), without the addition of carotene the percentage of germinated hemp pollen was 39%, the length of the pollen tubes was on the average 100 µ ; in the presence of carotene the percentage of germinated pollen was 53.5 and the length of the pollen tubes 312 µ . The lower the vitamin content, the sharper the reaction to the addition of carotene. Pollens rich in this vitamin stimulate the germination of pollen which contains small amounts of the provitamin if they are left to germinate together.

  Other vitamins (C₁, B₆, B₁, B₂, PP) also have an effect on the germination of pollen. Pollens of different species and also of different varieties of the same species do not give the same reaction to the addition of vitamins. For example, pollens of one variety of tobacco require 0.0002 mg of vitamin B₁, and pollens of another variety of the same plant require 0.005 mg per liter of the solution. Thirty one per cent of pine pollens germinated in a medium vitamin PP and in the presence of this vitamin 54% of the pollens germinated. About 10-12% of the pollen grains of one variety germinate in the presence of vitamin B₁ and in another variety--52% germinate (Polyakov, 1949).

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