HOME      AG LIBRARY      KRASILNIKOV TABLE OF CONTENTS


Part III, continued:

 

Microorganisms synthesizing biotic substances

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

  Wildiers (1901) showed the presence of activating substances in yeastcultures, These substances were called by the author bios substances, Fifteen totwenty 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 ofmany microbes.

  Investigations showed that biotic substances are synthesized by variousmicroorganisms--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 synthesizebiotic substances into auxoautotrophs and auxoheterotrophs. The former synthesizeall 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 synthesizeall the necessary biotic substances and therefore they cannot grow on vitaminlessmedia,

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

  Chemosynthetic bacteria are the most active in synthesizing bioticsubstances, They can grow in purely mineral, completely vitaminless media, beingcapable of synthesizing organic substances from atmospheric CO2. For example,thiamine, riboflavin, pantothenic acid, nicotinic acid, vitamin Be and some othercompounds were found in the culture of Thiobact. thioxidans (O'Kane, 1943).These bacteria and other similar chemosynthetic bacteria--the nitrifters and hydrogen-andmethane-oxidizing bacteria, synthesize their full quota of biotic substances. Withoutthis ability they could not have developed in mineral media,

  Bacteria incapable of CO2 assimilation, but growing wellin synthetic vitaminless media with organic carbon sources, are also able to synthesizebiotic 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 bacterialspecies among which were Ps. radiobacter, Bact. denitrificans, Bac. mycoides,Bac. subtilis, Bacterium sp., and others. Raznitsyna (1938) employing the coleoptilemethod has detected the formation of this compound by various representatives ofbacteria and mycobacteria. She divided microorganisms into three groups, accordingto 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 synthesizedby 99 cultures out of a total 150 studied, According to the authors, the most activeproducers of heteroauxins were bacteria and actinomycetes. Heteroauxin was foundin 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 otherscan be detected in bacterial cultures (Detinova, 1937, Leo and Burris, 1943, Jonesand 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 andLochhead (1951), Went and Wilson (1938, 1939). These and other vitamins were foundin cultures of Az. chroococcum, in various species and strains of the genusPseudomonas and in mycobacteria. In recent years vitamin B12 wasfound in many bacteria and especially in actinomycetes, Some of these organisms suchas Mycob. propionicum, A. rimosus, A. aureofaciens, and others, are employedin 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 bacterialcultures (Bac. subtilis, Bac. Mesentericus, Bac. mycoides, Bact. prodigiosum,various micrococci, and others).

  Landy, Larkum and Oswald (1943) found paraminobenzoic acid in culturesof 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 speciesof bacteria and actinomycetes.

  According to Schmidt and Starkey (1951), 99 species of bacteria andactinomycetes, out of a total of 150 species studied, synthesized heteroauxin. Twenty-twobacterial cultures out of a total of 75 cultures studied, synthesized vitamins onsynthetic media.

  We have studied 192 bacterial cultures isolated from different soilsof the USSR. These cultures were tested for their capacity to synthesize vitaminB1 and heteroauxin. The results are given in Table 42. It can be seenfrom the table that more than 50% of the bacteria studied synthesize vitamin B1and almost 40% synthesize heteroauxin.

Table 42
Biotic compounds in bacteria

Bacteria

No of cultures tested

Number of cultures synthesizing vitamin B1

Number of cultures synthesizing vitamin B2

Bac. subtilis

18

10

10

Bac. mesentericus

15

12

5

Bac. sp

13

3

1

Ps. flourescens

8

8

8

Ps. denitrificans

12

10

12

Ps. mycolytica

3

3

3

Bact. coli

2

0

0

Bact. proteus

2

0

0

Bact. liquefaciens

8

5

3

Bact. sp.

13

6

8

Rhizobium trifolii

15

10

0

Rhizobium phaseoli

15

12

0

Rhizobium leguminosarum

8

6

8

Az. chroococcum

16

16

10

Az. vinelandii

4

4

2

Mycob. album

12

7

6

Mycob. citreum

10

10

8

Mycob. rubrum

3

0

0

Microc. albus

3

0

0

Microc. flavus

5

1

1

Microc. aureus

6

0

0

  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 8days incubation the vitamin contents of the bacterial cells and of the culture filtratewere determined. The results are given in Table 43,

Table 43
The amount of vitamins synthesized by various bacteria, in µ g,
(after 8 days of growth)

Bacteria

Thiamine in 10 ml of medium

Thiamine in cells 1 g dry weight

Nicotinic acid in 10 ml of medium

Nicotinic acid in cells 1 g dry weight

Riboflavin in 10 ml of medium

Riboflavin in cells 1 g dry weight

Biotin in 10 ml of medium

Biotin in cells 1 g dry weight

Ps. aurantiaca

4.0

203

7.0

355

1.8

91

3.2

162

Ps. flourescens

0.2

23.3

4.4

511

0.14

162

0.18

20.9

Ps. radiobacter II

0.4

6.2

5.2

80.2

2.8

43

3.0

46

Ps. radiobacter III

0.3

13.2

4.0

176

3.6

158

0.6

26

Bact. herbicola

0.05

14.7

1.7

470

0.04

11.7

0.03

8.8

  Many fungi produce biotic substances. Thus, for example, thiaminewas 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, vitaminsC and K, and many others have been found in many fungi (Meisel, 1950).

  Rogosa (1943) studied 114 various yeast cultures such as Torulasphaerica (26 strains) T. cremoris (20 strains), Sacch. fragilis, Moniliapseudotropialis, Mycotorula lactis, Sacch. anamensis, Torulaopsis kefyr, Torula lactosa,Zygosaccharomyces lactis and others. In all the cultures they found vitamin B2in amounts ranging from 0.6 to 0.11 µ g per ml of synthetic medium.

  Biotic substances were also found in the mycorhiza fungi. Accordingto Schaffstein (1938), mycorhiza fungi of orchids synthesize growth factors requiredfor the normal growth of the host plant.

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

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

  Ondratschek (1940) found ascorbic acid, (vitamin C) in the secretionsof 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 butyricumrequires only biotin and synthesizes its other requisites. Some bacterial speciesrequire only thiamine or pantothenic acid. According to Burcholder, McWeigh and Mayer(1944) of 163 yeast cultures 87% required biotin, 35% thiamine and pantothenic acidand 12 % required only inositol.

  There are many bacteria which can synthesize only a fraction of avitamin's molecule. For example, some organisms synthesize only part of the thiaminemolecule, 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 ofa biotic substance (Meisel, 1950; lerumalimskii, 1949; Stephenson, 1951, and others).

  Thompson (1942) has shown that bacteria synthesize vitamins in amountsexceeding those required for their metabolism. The excess of vitamins is releasedinto 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 vitaminsby living organisms. The possibility in not excluded that some biotic substancesare waste products of growing cells.

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

  An idea on the quantitative aspect of the synthesis of biotic substancesby microorganisms can be obtained from the data in Table 44. These data have beencompiled from various sources.

Table 44
The synthesis of vitamins by microorganisms grown on nutrient media
(1 per µ g of, dry weight of cells)

Microorganisms

B1

B2

Nicotinic acid

Panto- thenic acid

B6

Biotin

Ino- sitol

Folic acid

Bact. aerogenes

19.9

154

630

780

26.8

47.9

1,400

105

Ps. flourescens

74

377

560

311

75.7

68.1

1,700

74.8

Bact. proteus

23

95

330

130

16.4

21.4

1,000

42

Clostr. butyricum

39.3

235

1,930

318

23.2

0

870

18.8

Az. vinelandii

96

351

593

184

--

4.2

--

--

Penicill chrysogenm

2.6

47

212

212

23.0

1.5

--

14.6

Sacchr. cerevisiae

360

42

1,000

100

100

1.2

5,000

31.2

Torula utiis

52.8

62

535

180

1.9

35

3,500

31.2

  According to West and Wilson (1938, 1939) there are 19.6 µ gthiamine and 0.37 µ g riboflavin per 1 g of dry weight of the root-nodule bacteriaof clover, grown on a synthetic medium. Clostridium (Clostr. butyricum) synthesizes0.9 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 perg 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 dryweight of cells.

  Considerable quantities of biotic substances are synthesized by manymycobacterial species. For example, Mycob. smegmatis synthesizes about 135µ g Oof vitamin B2 per ml of synthetic medium and 36 µ g perg of the dry weight of cells (Mayer and Rodbart, 1946). Forty to eighty µ gper 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 representativemicroorganisms. Their results are given in Table 45.

Table 45
Vitamin content of bacterial cells
( µ g per g of dry weight of cells)

Microorganisms

B1

B2

Nicotinic acid

Biotin

Peteroyl- glutamic acid

Pantothenic acid

Thiobact. thioparus

21

31

92

0.77

0.46

0.75

Thobact thiooxydans

23

60

15

0.64

1.89

57.0

Ps. pyocyanea

15

43

240

2.4

1.0

140.0

Ps. chroococcum

96

--

590

--

--

--

Propionibact. pentosaceum

6.4

--

--

--

--

93.0

Clostr. butyricum

9.3

55

250

1.7

0.5

92.0

Bact. proteus

21.0

--

250

3.4

4.2

100.0

Ps. flourescens

26.0

68

210

7.1

1.8

90.0

Bact. prodigiosum

27.0

35

240

4.1

3.2

120.0

  The above-given quantitative data are not strictly constant. Theymay vary, depending on the growth phase and culture conditions of the individualspecies of the bacteria, fungi or actinomycetes. In some cases old cells containless 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 culturesof Ps. radiobacter contain more vitamin B1 after 8 days growththan after 2 days growth.

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

  The intensity of the formation of vitamins by soil organisms is greatlyinfluenced by the symbiotic microbes. Some of them suppress vitamin synthesis andother stimulate this process. According to Smalii (1954), Azotobacter (Az. chroococcum)in pure culture synthesize 173 µ g of heteroauxin (per cell mass in 1 Petridish, on Ashby agar) and in the presence of the following microorganisms synthesizesthis 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 characterizea species Different strains of one and the same species differ markedly from eachother. For example, of more than 100 strains of Az. chroococcum which we isolatedin different soils and places in the USSR, some of them synthesized large quantitiesof heteroauxins and others synthesized only small quantities, or none at all. Formationof heteroauxin by the different representatives of these cultures in shown in thecoleoptile 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 andnot only for heteroauxin but also for biotin, thiamine, riboflavin, and other bioticsubstances.

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

  Only a few species of root-nodule bacteria are capable of synthesizingheteroauxin and even these are weak forms. We have investigated 12 species of root-nodulebacteria of clover, lucerne, kidney beans, vetch, Lathyrus vermus, lungwort,peas, Onobrychis, soya, lupine, acacia and astragalus. All these species either didnot synthesize heteroauxin at all, or synthesized it in small amounts only (Figure64).

 

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 strainssynthesized this compound in amounts able to give a barely perceptible coleoptilecurvature. Fifteen strains of Rhizobium trifolii, 8 strains of Rhizobiumleguminosarum and 15 strains of Rh. phaseoli were examined. In all casesthe picture was the same.

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

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

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

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

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

  Schmidt and Starkey (1951) have shown that if plant residues whichdo 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 decompositionof the residues by microbes. The increase in the riboflavin content of the soil isconcomitant with the intensification of microbial metabolism (Figure 65). The moreplant residues introduced into the soil, the more intense the microbial growth andthe 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.

 

Table 46
The formation of riboflavin in the soil during the decomposition of oat straw
(µg per 100 g of soil)
Accumulation of riboflavin in days: 0 days 1 day 3days 4 days 7 days 56 days
1.25 grams of straw applied 11 19 26 27 26 13
2.5 grams of straw applied 20 22 60 55 38 19

  Similar results are obtained if glucose or saccharose are introducedinto the soil instead of straw; the bacteria inoculated into the soil lacking thevitamins 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 vitaminB1, 300 g of vitamin B6 and 1 kg of nicotinic acid are synthesizedby microbes in the surface layer of one hectare of the fertile soils of the southernregions, during one season (9 months).

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

Table 47
The preservation of riboflavin and pantothenic acid in soil
(µg/100 g soil)

Vitamin

Amount introduced into the soil

Soil

Present after 0 days

Present after 1 day

Present after 2 days

Present after 3 days

Present after 6 days

Present after 21 days

Riboflavin

 

 

 

 

 

 

 

 

 

40

Sterile

38

33

--

40

34

34

 

40

Nonsterile

36

36

--

43

16

12

 

80

Sterile

69

68

--

81

67

64

 

80

Nonsterile

65

68

--

81

49

13

Pantothenic Acid

 

 

 

 

 

 

 

 

 

50

Sterile

34

34

35

35

 

 

 

50

Nonsterile

32

34

10

10

 

 

 

100

Sterile

72

77

80

73

 

 

 

100

Nonsterile

68

64

18

10

 

 

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

  If the microbial metabolism is artificially arrested, vitamins introducedinto contaminated soil persist for the same periods as in sterile soil. It was foundthat biotic compounds (vitamins and heteroauxin) persist in samples of dry soil takenfrom cultivated and fertilized fields, from 3 to 4 months to 4 years depending onthe 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 anotherroute are decomposed and synthesized de novo by microorganisms, Some vitamins disappearothers appear. There is a continuous turnover of these substances in the soil. Bioticsubstances can be found in the soil during the entire vegetative period an long asthe microbes live, reproduce and exhibit metabolic activity. The amount of the bioticsubstances is determined by the rate of their synthesis and introduction into thesoil and also by the rate of their destruction, or their stability, 

The effect of biotic substances on plants

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

  Different plants react variously to the addition to the substrateof growth factors and vitamins. Some respond by enhanced growth or by changes inthe course of biochemical processes, others react weakly and still others do notreact at all. This permits us to assume that the first produce only minimal amountsof the active substances which are insufficient for their normal metabolism, thesecond synthesize them quite actively but in amounts still insufficient to satisfyall their needs, and the third synthesize them in adequate quantities.

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

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

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

  The plants' requirements for vitamins and auxins has been thoroughlystudied in experiments with isolated organs and tissues, and especially with isolatedroots.

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

  Investigations show that roots of different plants demand differentgrowth factors. For example, roots of flax require vitamin B1, roots ofpeas, horse-radish, lucerne, clover and cotton require vitamins B1 andB6; 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 syntheticmedia, synthesize all the required growth factors. Some of them synthesize them inamounts sufficient for their normal growth, others form too little. The first growwell 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 requirementsof 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 isolatedroots. The roots of flax in the presence of vitamin B1 elongated by 185mm. and in its absence by 31 mm in one week. The roots of flax are calculated tosynthesize vitamin B1 at a rate of 0.02 µ g per week. Their vitaminB1 requirement for normal growth is 2 µ g, i.e., 100 times more thanthey synthesize.

  The roots of white clover grow well in a medium containing vitaminsB1 and PP*. *[ The correct designation of this vitamin is unclear.] Theyincrease in length with each successive transfer into a fresh nutrient medium. Inthe first 5 weeks the roots elongate by 84 mm, the increment in the next 5 weeksamounts 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. Theincrement becomes uniform upon subsequent transfer amounting to about 22 mm per week.

  The roots of sunflower in the absence of the vitamin complex or inthe presence of only one of the vitamins PP or B6 cease to grow after7 consecutive resowings. Roots which were supplied with all three vitamins, PP, B1and B6 grew well for along period of time allowing for many transfersinto fresh media. In the first five weeks the increment was 74 mm, in the next 5weeks it amounted to 96 mm, in a further 5 weeks--120 mm, and in the following fortnight--150mm.

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

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

  Went, Bonner and Warner (1938) had shown that thiamine stimulatesthe growth of roots of peas, lemons and camellia. The results were more markedlypronounced when a mixture of thiamine and heteroauxin was employed. Positive resultswere also obtained in these cases with a mixture of vitamin B1 and indoleaceticacid (Grebenskii and Kaplan, 1948) and also with vitamin K, biotin and pantothenicacid with biotin (Scheurmann, 1952).

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

  In some cases roots of certain plants required only parts of the vitaminmolecule, 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 rootsof which can grow on nutrient media. There are 22 such species. Roots of 27 speciescould 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 donot grow in the same manner as when attached to the plant. They grow in length andbranch, but do not get thicker or if they do thicken, then only very slightly. Theactivity 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 certainvitamins added to the substrate. For example, the growth of pea seedlings separatedfrom the cotyledons considerably increases in the presence of thiamine and biotin(Kögl and Haagen-Smit, 1936). Pantothenic and ascorbic acids also act favorablyon pea embryos (Bonner T. and Bonner H. , 1948).

  Plant embryos do not synthesize biotic substances, they utilize thefood reserves present in the seeds. Even the green sprouts of many plants in theearly 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 vitaminswhile the nonripe embryos require vitamins PP, B1, B6, C andothers.

  Pantothenic acid also has a favorable effect on lucerne sprouts. Treatingpea 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 thepresence of an 0.01 % solution of vitamin PP and, moreover, the formation of rootsand the growth of aerial parts is more intense (Flerov and Kovalenko, 1947).

  The presowing treatment of cotton seeds with vitamins B1and 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 ascompared to 45 % in the control. The length of the sprouts in the former case wason 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 aftertreatment 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 masswas greater by 39%, and the yield of seeds was 28% higher then that of the controlplants (Dagis, 1954).

  Bonner et al. reached the conclusion that the lower the vitamin contentof plant leaves, the stronger they react to the addition of these substances. Accordingto them, peas and tomatoes, contain 13-18 µ g of vitamin B1 per kgof dry leaves and do not react to its addition. Cabbage, cosmos, Japanese camelliaand others contain small amounts of vitamins in their leaves and react positivelyto 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 smallamount of vitamins in their leaves and react less to their addition than plants withhigher 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 daystwice as much as the controls. The application of low concentrations of vitamin B1to poppies increased the weight of their bolls as well as the crop in general. Applicationof vitamin B1 together with water had a favorable effect on the growthof spinach. The weight increment during the 63 days of the experiment exceeded manytimes that of the control plants (Table 49).

Table 49
Dry weight of spinach leaves (in mg) at the end of 63 days of the experiment, after 13 irrigations with a solution of vitamin B1
Treatment

1*

2*

3*

4*

5*

6*

7*

8*

9*

Control

32.4

58.1

56.2

35.8

9.6

--

--

--

--

Vitamin B1

28.5

103.9

255.6

390.0

504.1

534.9

375.9

205.0

45.7

*This is unexplained in Russian text. Probably refers to position of leaves on plants.

  Denisov introduced vitamin B2 into a substrate where eggplants were grown and obtained a marked increase in yields. After 77 days growththe control plants had stems 7.1 cm long, the weight of the tops was 48 5 g and theweight of the roots 12.5 g. The corresponding figures for plants grown in the presenceof 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 underthe influence of vitamin PP. Lemon seedlings react markedly to the addition of thisvitamin (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 theirexperiments with a Bukhara almond. The plants were sprayed with an aqueous solutionof 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 controlplants.

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

Table 51
The effect of vitamins on the growth of the orchid Thunia marschaliana Rchb. f.
(according to Henrikson, 1951)

Vitamins

Height of plants, mm

Number of leaves

Length of roots, mm

Dry weight, mg

Control

48.0

5

60.5

30.8

B1

83.5

6

92.5

59.1

B6

48.0

6

58.5

59.1

C

55.5

6

55.5

30.1

PP

101.0

8

176.5

86.6

  Rakitin and Ovcharov (1948) employed vitamin PP and adenine for increasingthe 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 cottonyield (raw material) was considerably higher than that of the controls. Similar datawere obtained by Zakhar'yants, Gorbacheva and Zglinskaya (1950). They sprayed cottonplants 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 theB-group suppress the growth and diminish the crop of plants. Carotene suppressesthe growth of safranin which is itself rich in carotene. Vitamin K suppresses thegrowth of fungi, some bacteria and the roots of higher plants. Vitamin PP antagonizesthe action of vitamin K. Vitamin E, according to Schopfer (1950), arrests the growthof certain plants. The height of plants in the control was 35.75 cm and in the presenceof 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 thiaine and yeast extract, Such procedure markedly stimulated the growth and increasedthe crops, the seeds too were larger.

  Söding, Bömke and Funke (1949) obtained 30% higher yieldsof 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 thegrowth of lucerne under sterile conditions in sand cultures, in a medium which didnot contain nitrogen. Pantothenic acid was added in high concentrations. Plants underthese conditions grew in the presence of pantothenic acid ( in high concentrationsof pantothenic acid) much better and the yield was higher.

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

  Shavlovskii (1954) tested the effect of pantothenic acid, vitaminB1, nicotinic acid and vitamin B6 on the growth of lucerne.The latter was grown on agar medium under sterile conditions for 30 days. The resultsare given in Table 52.

Table 52
The effect of vitamins on the growth of lucerne
(vitamin concentration in the medium = 0. 1 µ g/ml)

Vitamins

Dry-mass weight of 20 plants in mg: Tops

Dry-mass weight of 20 plants in mg: Roots

Dry-mass weight of 20 plants in mg: Total

Control (without vitamins)

38.6

8.0

46.6

Pantothenic acid

36.4

11.6

48.0

Vitamin mixture

37.2

12.2

49.4

  Analogous experiments were carried out by Shavlovskii with buckwheat.The plants were grown in sand wetted with the nutrient solution of Hellrigel. containing1 µ g of the vitamin per ml. Other containers were supplemented with yeast extractand vitaminless casein hydrolysate. In one series of experiments the bacterial cultureof Ps. aurantiaca--vitamin producers were introduced. Plants were grown for2 days and then analyzed. The results are given in Table 53.

Table 53
The effect of biotic substances on the growth of buckwheat

Biotic compound

Dry mass weight of 10 plants in mg: Cotyledons

Dry mass weight of 10 plants in mg: Stems

Dry mass weight of 10 plants in mg: Roots

Dry mass weight of 10 plants in mg: Whole plant

Control without vitamins

73.0

64.0

32.0

168.0

Bacteria Ps. aurantiaca

76.0

64.0

41.0

181.0

Vitamin B1

82.0

63.0

39.0

184.0

Vitamin B12

79.0

64.0

32.5

175.5

Vitamin mixture

80.0

65.0

36.0

181.0

Yeast extract 0.01%

80.0

66.0

40.0

186.5

Yeast extract 0.1%

90.0

65.0

40.0

195.0

Casein hydrolyste 0.1%

80.0

66.0

43.0

189.0

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

  The biological role of vitamins has been little studied, but, accordingto the available data, it is important. It is well known that many of them are componentsof various enzymatic systems. The so-called coenzymes which enter into chemical interactionwith the substrate include many vitamins, It has been found experimentally that vitaminB1 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 varioustransformations of carbohydrate compounds, including pyruvic acid, are not feasible.The latter is the key intermediate in the metabolism of living cells, which linksthe metabolism of carbohydrates, proteins and fats.

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

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

  Vitamins also play a considerable part in the formation and transformationof proteins. It has been shown that vitamins B2, B6, B12,PP and H participate in the formation of amino acids and their transaminations. Theshortage of vitamin B6 leads to a decrease in the formation of amino acidsfrom organic acids and ammonia. Vitamin Be takes part in the formationof amino acids from organic acids and ammonia. Transamination, i.e., transfer ofan amino group (NH2) from one acid to another, takes place in the presenceof vitamin B6.

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

  Vitamins play an immense role in respiration. It was shown that enzymesparticipating in respiration consist of proteins and a coenzyme. The latter consistsof vitamin B2 and phosphoric acid. Vitamin B2 in enzymaticsystems plays a role in oxidation-reduction processes. Folio acid is of great importancein respiration. The germination of seeds and the respiration of sprouts increasesunder the action of this acid (Stephenson, 1951, Schopfer, 1943, Zeding, 1955).

  Growth stimulators--auxins and heteroauxins--have a positive effecton the colloidal and chemical properties of protoplasm. According to some authors,they participate in the general metabolism of the cell as separate components. Byincreasing the metabolism they influence the growth of the cells of the aerial partsand especially of the roots. Under the influence of heteroauxin the influx of plasticsubstances 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 cellsof the latter. The activity of peroxidase is also increased and the tissues are betterhydrated (Maksimov, 1940, Turetskaya, 1955, Zeding, 1955, and others). The actionof 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 otherbiotic substances. Data exist which show that heteroauxin stimulates the formationof auxins, (Zeding, 1955).

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

  Vitamins have a favorable effect on the fertilization of plants. Itwas found that the sexual organs are rich in vitamins especially the pollen. Forexample, 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 itsactivity. Processing of such pollens with carotene increases its capacity to germinate.Thus, according to Lebedev (1952), without the addition of carotene the percentageof germinated hemp pollen was 39%, the length of the pollen tubes was on the average100 µ ; in the presence of carotene the percentage of germinated pollen was53.5 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 vitaminstimulate the germination of pollen which contains small amounts of the provitaminif 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 speciesand also of different varieties of the same species do not give the same reactionto the addition of vitamins. For example, pollens of one variety of tobacco require0.0002 mg of vitamin B1, and pollens of another variety of the same plantrequire 0.005 mg per liter of the solution. Thirty one per cent of pine pollens germinatedin 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 vitaminB1 and in another variety--52% germinate (Polyakov, 1949).




HOME      AG LIBRARY      GO TO PART III. section 5