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Part IV, continued:

 


  Winter and Willeke (1951, b) have shown that in tissues of lettuce, penicillin may accumulate up to 500 units/g, and streptomycin, up to 100 units/g and cause no noticeable pathological phenomena. When the penicillin concentration in the tissues reaches 1,000-2,000 units/g, the plant suffers from poisoning.

  Brian, Wright, Stubbs and Way (1951) point out that considerable concentrations of griseofulvin in the tissues of oat and lettuce brought about no poisoning effect.

  If the concentrations of the antibiotics in the substrate are very low, the plants may accumulate them in their tissues. For example, when the concentration of penicillin in the medium is 50 units/ml, up to 80 units/g accumulates in the roots of or wheat and corn; when the concentration of grisein is 10 units/ml in the medium, up to 20-50 units/g and more are accumulated in the roots of peas.

  Pramer (1953-55) tested different antibiotics--streptomycin, aureomycin, chloromycetin, terramycin, neomycin, etc. According to his observations, different substances enter the plants and saturate their tissues to various degrees. In experiments with cucumber seedlings, the most highly absorbed drug was streptomycin, At a concentration in the solution of 500 µ g/ ml, its accumulation in the tissue reaches 100-150 µ g/ g, 18 hours after treatment. Streptomycin penetrates less readily and in smaller quantities the tissues of tomatoes and kidney beans. The author notes that sometimes the concentration of streptomycin in leaves is higher than that in stems and roots.

  Chloromycetin penetrates the cucumber seedlings much more weakly than does streptomycin and reaches a concentration of 20-50 µ g/g. Aureomycin, terramycin and neomycin do not penetrate this plant at all.

  Pramer (1954), investigated the absorbability of streptomycin, chloromycetin and penicillin into the cells of algae Nitella clavata and found that after the algae were immersed for 12 minutes in a streptomycin solution which contained 8 µ g /ml, the algae's cell sap contained the same concentrations of antibiotic as that of the external solution. After immersion in the same solution for 18.5 hours the concentration of streptomycin in the sap was 7 times higher than that of the surrounding solution.

  Chloromycetin penetrated the cells of the algae with difficulty. Only after 24 hours was it detected in the sap. Penicillin was not detected at all in the cells of the algae after 25 hours of immersion in a solution with 25 µ g/ml antibiotic concentration. The author believes that penicillin penetrates the cells quickly, but is immediately oxidized.

  The concentration of antibiotics in plant tissues depends not only on the amount of the substances entering but also on the rate of their disappearance, or the time of their preservation.

  Antibiotics are known to be preserved in the body of animal organisms for a short time only. They are excreted from the body during the first hours after their entrance, which complicates the work with antibiotics in hospitals. Only with the aid of special substances--prolongators--does one succeed in keeping the antibiotic in the animal or human organism.

  In plants the antibiotics which are introduced are preserved for much longer periods of time. These very same substances -- penicillin, streptomycin, globisporin, aureomycin, etc are preserved within the plants for several days or even weeks. For example in tissues of sweet cherry, penicillin is preserved for 4 days and in tissues of the apricot tree--16-17 days (Table 131).

Table 131
Time of preservation of penicillin inside woody plants

Plants

Age of plant, years

Antibiotic indroduced, in units

Time of preservation in days

Location where experiments were carried out

Cherry

8

2,400,000

8

Moscow, Central Botan. Garden Ac. Sci, USSR

Apricot

5

2,200,000

16

Moscow, Central Botan. Garden Ac. Sci. USSR

Peach

8

4,800,000

15

Crimea, Nikitskii Botan. Garden

Apricot

7

6,000,000

17

Crimea, Nikitskii Botan. Garden

Sweet cherry

7

1,450,000

4

Crimea, Nikiskii Botan. Garden

  In grassy plants antibiotics are preserved for a similar length of time.

  Grisein (No 15) when introduced into tissues of the cotton plant and peas, is preserved there for 10-20 days (Table 132).

Table 132
Time of preservation of the antibiotic grisein No 15 in plants
(unit/g of tissue)

Time of analyses, number of days after introduction

Cotton, roots

Cotton, leaves

Peas, roots

Peas, leaves

Initial amount

350

100

180

80

1

200

50

120

60

2

150

30

100

40

3

100

20

70

30

5

80

10

30

10

7

50

8

10

5

10

30

5

10

5

20

10

0

0

0

30

0

0

0

0

  Askarova (1951) and Mirzabekyan (1953, 1955) found antibiotic substances inside the cotton plant 20 days after their introduction.

  Brian et al., (1951) detected griseofulvin in tissues of lettuce and oats during 3-4 weeks.

  Antibiotics first disappear from the aerial parts of the plants and later from the root system.

  Analyzing a nutrient solution in which plants saturated with antibiotics have been kept, we could establish that these substances were excreted by the roots. However, the amount of the substances excreted was much smaller than that of the plant. If in the tissues of one pea plant there were about 4,000 units of penicillin then about 600 units were excreted into the solution.

  In the experiment with streptomycin, single pea plants were immersed for one day in streptomycin solution. The number of units of the antibiotic which were absorbed from the solution were precisely determined, and the plant was removed from this solution. The roots were washed with water and immersed in Hellriegel' s nutrient solution which did not contain antibiotic. After certain time intervals the amounts of the antibiotic in the solution and in the plant tissues were determined. The results are given in Table 133.

Table 133
Excretion of streptomycin from pea tissues into solution

Times of analyses, number of days after introduction

Units in solution

Units inroots

Units in stems

Units in leaves

Units in in the whole plant

Initially

0

350

45

80

1,707

3

120

270

20

40

1,123

5

240

150

20

20

767

10

320

20

5

5

105.5

15

350

0

0

0

0

  In these experiments one pea plant absorbed 1,800 units, after 10 days only 105 units were found, and after 15 days nothing at all was left. During this time only 350 units were excreted into the solution by the roots. Therefore, we assume that the remaining 1,357 units of antibiotic were assimilated by the tissues as sources of nutrition and underwent biochemical changes.

  Comparing the degree of absorption of the antibiotic with the nature of the distribution of the latter in the plant, and with the time of its preservation in the tissues, one may conclude that there exists a direct connection between these phenomena. The more the antibiotic was absorbed, the sooner it was found in the leaves and upper branches, and the longer it was preserved there.

  However, this is not always so. Quite often, upon intense absorption of an antibiotic the latter is not detected in the tissues or is found there in very small quantities. For example, maple and bird cherry under similar conditions absorb the same amounts of penicillin, but in the bird-cherry tree it penetrates into the upper parts and reaches the leaves, while in the maple it is found neither in the leaves nor in the branches. In the apricot, peach, sweet cherry and apple trees penicillin may be detected in the leaves upon introduction of 350-500 thousand units per tree while in maple, ash tree, lime tree and acacia it cannot be detected even when it is introduced in considerably larger quantities.

  We introduced 7-15 million units per tree of globisporin and penicillin into the trunks of a 10-year-old birch and a 7-year-old willow--calculated on the basis of 300-600 units per 1 g woody mass. After 36 hours penicillin and globisporin could be detected in the leaves of the willow; the former was 1.5-2 times more concentrated than the latter (Table 134). In birch the antibiotic was not found either in the leaves or in the branches. However, traces of the antibiotic were detected in the wood of the trunk at a distance of not more than 10-30 cm. upward and downward from the point of introduction.

Table 134
The uptake and distribution of penicillin and globisporin in tissues of birch and willow (solutions introduced: penicillin--15, 000 units/ ml globisporin--10,000 units/ml)

Type of Antibiotic/Type of tree

Amount of anti- biotic solution introduced

Total units per plant

Units per 1 g of wood mass

Anti- biotic found after 10 hours

Anti- biotic found after 20 hours

Anti- biotic found after 36 hours

Anti- biotic found after 48 hours

Anti- biotic found after 72 hours

Anti- biotic found after 120 hours

Birch

 

 

 

 

 

 

 

 

 

Penicillin

1,000

15,000,000

600

0

0

0

0

0

0

Globisporin

800

8,000,000

250

0

0

0

0

0

0

Willow

 

 

 

 

 

 

 

 

 

Penicillin

850

13,600

500

0

trace

20

30

20

15

Globisporin

680

6,800,000

300

0

0

10

15

15

10

  The absence of antibiotics in the leaves and branches of birch, maple, lime tree and other plants may be explained by their inactivation or by the adsorption by the tissues adjacent to the point of introduction, and, in certain cases, also by the weak uptake of the solution. The last explanation does not apply in the case of the birch. As is seen from the table, the birch absorbed more penicillin and globisporin solution than the willow, but nevertheless, the antibiotic was not found either in the leaves or in the branches.

  Investigation of the causes of this phenomenon has shown that the wood of the trunk and branches and the leaf mass of birch possess a clearly expressed inactivating and absorbing capacity in relation to the antibiotics tested. By specially devised methods we have established, that one gram of wood of the trunk of birch absorbs 18,000 units of penicillin and fully inactivates 6,000 units; it absorbs 6,000 units globisporin and inactivates more than 3,000 units. A ground mass of green leaves absorbs 10,000 units penicillin and inactivates 8,000 units; it absorbs 6,000 units globisporin and inactivates 5,000 units. In other words, the wood and especially the leaf mass of the indicated plants almost completely inactivate the absorbed antibiotics--penicillin and globisporin. In order to detect the antibiotics in the given tissues, it is essential that they be administered in a higher concentration (more than 6,000 units globisporin and more than 8,000 units penicillin per g).

  Plant tissues probably inactivate all other antibiotics which enter them. We tested streptomycin, penicillin, aureomycin and certain crude preparations of actinomycetal origin on various plant tissues: apples, lemons, peaches, cherries, etc. In all cases a different degree of inactivation was observed. Thus, tissues of lemon saplings (3-5 years old) inactivated aureomycin within the limits of 50--100 units/g, the leaf tissue was a stronger inactivator than the tissue of the trunk. The tissues of the apple tree and even more so, those of ornamental woody plants inactivated aureomycin to the extent of 200-500 units/g.

  Inactivation of the crude preparation No 399 (from strain 399) in our experiments, was as follows:

Tissue of the lemon-tree trunk; 70 units/g
Tissue of the lemon-tree leaves; 160 units/g
Tissue of the pear-tree leaves; 220 units/g
Tissue of the apple-tree leaves; 180 units/g

  Antibiotic substances may be introduced into the plant through the aerial parts, not only as chemically pure preparations, but also in their crude state, in the form of a culture fluid which is diluted with water to a certain concentration. We introduced the crude antibiotics via the trunk and through the leaf surface of woody plants and grasses. Plant seeds were also soaked in crude substance seeds of wheat, clover, peas, etc.

  Antibiotic substances introduced in the form of culture liquid are distributed in the same manner as are chemically pure preparations, but at lower rates.

  If seeds treated with antibiotics are immediately germinated, these substances may be detected in the seedlings. This translocation of the antibiotics from the seeds to the seedlings was observed by us in the cotton plant, peas and wheat. Special analyses have shown that the antibiotics completely permeated the cotyledons of the leguminous plants, as well as the endosperm of the cereals. Such a saturation of the food reserves with antibiotics--penicillin, streptomycin, grisein and certain other substances, does not cause any harm to the seedlings. The latter develop normally and utilize the reserves of the endosperm or the cotyledons exactly as the control seedlings but only if the antibiotic is not toxic.

  Antibiotics introduced into plants have an antimicrobial action. If plants are artificially infected with a phytopathogenic form of bacteria or fungus, and a corresponding antibiotic is employed, the disease will not appear or will be weaker than it is in the control. We have introduced many nonpathogenic bacteria inside plants--Bact. coli, Bact. prodigiosum, Bact. album, Ps. fluorescens, Ps. sp., Rhizobium trifolii, etc. They all died much more rapidly in the tissues of plants to which antibiotics were introduced. For example, the root-nodule bacteria of clover, Bact. coli and Bact. prodigiosum, introduced into the stem of peas or kidney beans, die there after 20-30 hours and later, while the plants treated with streptomycin showed no bacteria after only 2-6 hours. The phytopathogenic fungus Fusarium sp. spread on seedling of pine, grew well on them, penetrated the inside and caused their death after several days. On plants that were treated with the corresponding antibiotic (No 121), the given fungus did not grow, and the growth of the plant was normal. There are many other observations which demonstrate the antimicrobial action of antibiotics inside plant tissues.

  The antibiotics used should not be toxic to the plants. It is known that among the antibiotics there are various preparations, some of which are very toxic and cause the poisoning of certain tissues or of the entire plant. To such antibiotics belong: gramicidin, mycetin, clavacin, catenulin, magnamicyn, etc. Clavacinan antibiotic produced by the fungus Asp. clavatus, inhibits the growth of cereal roots at a dilution of 1:1,000,000 (Wang 1948). Other antibiotics--penicillin, streptomycin, grisein, terramycin, etc--may for all practical purposes be considered nontoxic. They may accumulate in tissues in large quantities, do not cause any disturbances, and, at certain concentrations, even stimulate the growth of plants (Barton and Mac Nab, 1954; Askarova, 1951), Scheffer and Kloke (1954) introduced antibiotics into soil in which they later cultivated plants. Only very high concentrations of the antibiotics caused the inhibition of the growth of barley and rye.

  There is no need to introduce over-large amounts of antibiotics in treatment. The antimicrobial doses of the antibiotics of this group are considerably lower than the doses which cause the poisoning of the plants. For example, penicillin inhibits the growth of bacteria in wheat tissues at a concentration of 3-10 units/g, while the plant can withstand a dose of more than 1, 000 u/ g. Streptomycin and globisporin inhibit growth of bacteria in plants at concentrations of 5-10 units/g while the plants can withstand a dose of more than 500 units/g, etc.

  There are many antibiotics that occupy an intermediate position in relation to their toxicity. Such antibiotics can also be successfully used in the healing practice. Griseofulvin is one of them. Its therapeutic dose is 5-10 µ g/g. A dose of 20 µ g/g is toxic for wheat and causes a burn and the swelling of the roots (Stokes, 1954).

  One should also emphasize another feature of the action of antibiotics, namely their ability to inactivate toxins formed by fungi and bacteria. Inactivation of toxic substances by products of microbial metabolism was mentioned before (Krasil'nikov, 1947 a). It has been shown, that the toxic effect of gramicidin can be eliminated by neutralizing it with the metabolic products of bacteria. Actinomycetes inactivated the toxin which was formed by the sporeforming bacteria, Bac. subtilis and Bac. mesentericus.

  It is known, that in many infections (if not in all) the plants suffer from poisoning by the toxins which are produced by microbes developing in the affected tissues.

  Therefore, by selecting appropriate microbes these toxins can be rendered harmless and thus the poisoning of the plant can be prevented. Clover seeds saturated with bacterial toxin did not germinate in our experiments, or germinated to a limited extent; their seedlings lagged in growth and soon perished. When the poisoned seeds were treated at once with antitoxin, the percentage of germinating seeds increased considerably and the growth of these seedlings was only slightly less than that of the controls (Figure 97). Toxin, which was mixed with antitoxin in a certain ratio had no toxic effect at all.

 

Figure 97. Antitoxic effect of metabolites (antitoxin) of bacteria on clover seedlings, poisoned with bacterial toxin:

a--control; seeds of clover were not treated with toxin before sowing; growth normal; b--seeds treated with toxic product, formed by bacterial inhibitors--there is no germination or a weak one; seedlings soon perish; c--seeds treated with the same toxin and then with the antitoxin substance produced by bacteria.

 

  The inactivating effect of certain actinomycetes was observed by us in experiments with toxins formed by the fungus Botrytis cinerea. This fungus grows abundantly on leaves of certain plants--birch, oak, etc which are wetted by the carbohydrate excretions of aphids. The toxic substances formed by the fungus affect the tissue of birch leaves. Toxins of the given fungus were obtained by us in a culture in a nutrient medium with honey produced from insects. Its application to the surface of a birch leaf causes burn and necrosis of the tissue with subsequent yellowing and death of the leaf.

  An antitoxin against this toxin which was formed by an actinomycete antagonistic to Botrytis cinerea was found. The addition of a certain amount of antitoxin to the toxin neutralizes it, and the mixture obtained is rendered nontoxic for birch. This antitoxin is also beneficial when first symptoms of poisoning appear. The process of wilting and formation of necrotic patches stops and the leaves recover from the injury and continue to develop normally.

  The antitoxic action of actinomycetes, or more exactly, the action of their metabolic products, was observed by us in experiments with toxins formed by the fungi Fusarium vasinfecturn, Fusarium sp., Trichothecium etc.

  The phytopathogenic fungus--Deuterophoma tracheiphila causes the poisoning of citrus plants by its metabolic products. An antagonist (A. griseus) of this fungus was found among the actinomycetes, which produces antitoxic substances and inhibits the growth of the fungus. The antibiotic grisein, obtained from a certain actinomycete, also had suppressive effects on this pathogen, which is the cause of a disease of citrus plants.

  It may be assumed that for any toxin of microbial origin an antitoxin can be found.

  Among the microbes there are many species which form strong poisons not only for plants but also for animals and human beings (botulin, tetanus toxin, etc). Under natural conditions these toxins are inactivated by other microbes, which produce antitoxins. It is tempting to use these antitoxins against food poisoning and other toxicoses of man and animals.

  As is seen from the above-mentioned data, antibiotic substances fulfill all the requirements demanded of healing substances in plant breeding.

  The possibility of using antibiotics for curative purposes was also proven by indirect laboratory and field experiments.

  The first experiments in this direction were performed with crude antibiotic substances, obtained from bacteria and actinomycetes (Krasil'nikov, 1947 a). More fundamental and systematic studies were performed with chemically purified substances. Antibiotics were employed in the struggle against infections of woody and grassy plants (Krasil'nikov, Mirzabekyan and Askarova, 1951; Askarova, 1951; Mirzabekyan, 1952). Mirzabekyan employed the specially chosen antibiotic, grisein, in treatment of apricot and peach trees suffering, from "bacterial wilt." This disease is caused by Bact. armentaca. It is expressed in the withering of the crown first, and then of the whole tree.

  At first the experiments were performed on young one- to two-year-old saplings of apricots and peaches. They were artificially infected with a culture of Bact. armeniaca and a few days later, treated with antibiotics.

  An aqueous solution of the preparation was introduced into the leaf surface by wetting it. In all cases where the plants were treated with antibiotic immediately after inoculation, the disease did not appear. In cases where the treatment was started after a delay and when the symptoms of the disease, the wilting of leaves, were already apparent the disease stopped developing, leaves and branches recovered, and the plant continued to develop normally.

  A hundred per cent of plants, that were not subjected to treatment, were infected and perished (Figure 98).

 

Figure 98. Curative effect of antibiotics. Apricot seedlings infected with Bact. armeniaca:

a and b--plants not treated with antibiotic; c--plants treated with antibiotic; d--control plants (noninfected).

 

  Experiments with fruit-bearing, 15- to 20-year-old plants were performed in the experimental fruit section of the Academy of Sciences of the Armenian SSR and in one of the fruit farms of Armenia.

  The antibiotic grisein was introduced into the stem and was also sprayed on the crown. The process of drying ceased after the treatment. The leaves and branches recovered and continued to grow normally (Figure 99). In cases where the injury was a severe one and where there were dying branches, an effect was also noticed. The antibiotic stopped the further spread of the disease and new sprouts appeared on the still living parts.

 

Figure 99. Curative effect of the antibiotic grisein in "bacterial wilting" of apricots:

a--a tree not treated with the antibiotic; b--a treated tree.

 

  Positive results were obtained upon the use of antibiotic substances in the struggle with "malsecco" of citrus plants under laboratory conditions. The disease known as "malsecco" is caused by the fungus Deuterophoma tracheiphila.

  Young lemon trees artificially infected with the fungus easily succumbed to the "malsecco" disease. In the struggle with this disease antibiotics were selected which were later tested on experimental plants. The antibiotic solutions were introduced into the trunk and through the leaf surface.

  Among the preparations tested, grisein had a curative effect. The plants recovered quickly or did not get sick at all, while the control plants which were not subjected to the treatment, died,

  In order to sterilize the grafts of lemons which were used as grafting material Mirzabekyan (1955) saturated them with an antibiotic solution. Specialists have found, that the infection is introduced with the grafting material, In the laboratory experiment it was shown quite feasible to employ antibiotics in the practice of grafting plants; grafts treated with streptomycin or grisein were sterile.

  Among the grassy plants, antibiotics were most frequently used with cotton cultures, affected by gummosis. This is a widespread disease and causes great losses to agriculture. It is caused by the nonsporeforming Ps. malvacearum, which is distributed and carried by the seeds. These often harbor the bacteria internally, thereby considerably complicating the struggle against infection.

  Askarova (1951-52) preliminarily chose a number of antibiotics which inhibit Ps. malvacearum and she discovered that some of them (Nos 73/20, 160, 114, etc) penetrate the seeds of cotton freely and kill the bacteria which cause gummosis. These substances therein saturate the tissues of the entire seed and embryo but do not harm them. The ability of such seeds to germinate does not decrease and in some cases (preparations 160, 265) it even increases. At first the experiments were performed in growth containers and later on experimental plots and on industrial fields. Only crude antibiotics in the form of culture fluid were employed. The activity of this fluid was 1,000-2,000 units/ml.

  The results were positive. Seeds treated with antibiotics germinated better and their shoots were taller and healthier. There were fewer diseased plants at the early stage of growth and at the end of vegetation, than in the control (Table 135).

Table 135
Effect of antibiotics on the appearance of gummosis in the cotton plant
(experiment under field conditions on small plots)
(after Askarova 1951)

Conditions of experiment

Morbidity, % in the cotyledon stage

Morbidity, % in stage of boll formation

Control (no treatment of seeds)

72.4

19.1

Seeds treated with preparation No 114

8.1

0.0

Seeds treated with preparation No 117

10.2

0.4

Seeds treated with preparation No 86

18.4

2.0

 

 

 

 

Yield of seed cotton

 

From control plot

1,640 kg

 

From experimental plot, prep. No 114

3,040 kg

 

From experimental plot, prep No 160

3,480 kg

 

From experimental plot, prep No 86

2,250 kg

 

  In the experiments, seeds treated with antibiotics germinated earlier which was quite distinctly reflected in the subsequent development of the cotton plant (bud formation, flowering, opening of the bolls); the vegetation time of the plants was shortened by 8-10 days.

  Similar results were obtained in experiments performed under industrial conditions. Pretreatment of seeds with antibiotics 105 and 114 before sowing lowered the disease incidence of cotton-plant gummosis 5-6 times and because of this a higher cotton crop was obtained. For example, in the kolkhoz "Kzyl-Argin" (Tashkent Oblast') twenty-five per cent of the control cotton plants were affected by gummosis and on the treated fields the morbidity decreased to 5.3%. The control cotton plants yielded 24 centners/ hectare of seed cotton and the experimental plants--30.6 c/ha (Askarova, 1951-1952).

  Similar experiments were performed by Mirzabekyan (1952-1953) in Armenia. She employed grisein No 15 in the form of partially purified raw material in treatment of seeds. The incidence of the gummosis disease of the cotton plant decreased by 67-84%,

  Of interest were the experiments of Askarova (1952) against the secondary gummosis infection of the cotton plant. In cotton-growing practice one often observes a secondary massive infection of a given culture by gummosis during vegetation.

  This is often facilitated by rainfall. Under conditions of a field experiment and on the kolkhoz fields, application of antibiotic substances during this secondary infection also yields positive results.

  Bel'tyukova (1951) disinfected seeds with an antibiotic microcide before sowing. The incidence of infection of the plants with pathogenic bacteria decreased considerably after such treatment.

  Bushes of garden rose affected by mildew were specially treated by us with an especially chosen antibiotic of actinomycetal origin. After the leaves were washed three times with a solution of the crude antibiotic, the symptoms of mildew began to disappear and after some time the leaves acquired a normal or almost normal appearance. (Figure 100).

 

Figure 100. Treatment of bushes of garden roses, affected by mildew:

A--bushes not treated with antibiotic; the leaves are covered with a white coating of fungus; B--bushes treated with antibiotic; green leaves, free of fungus.

 

  A positive effect of antibiotics was observed in all cases where treatment was started in the early stages of the disease.

  In strongly affected bushes the treatment had only partial results: the mildew cover disappeared, but the leaves acquired a brownish-green color, which either disappeared later, or as more often occurred, remained until the end of vegetation.

  Positive results with antibiotics were obtained by Protsenko in floriculture, Gurinovich in market gardening and Afrikan and others in experiments with field crops and vegetable cultures.

  Recently, foreign investigators extensively investigated antibiotics in their a struggle against plant diseases under conditions of production, in orchards, gardens and fields. For this purpose special preparations of antibiotics are produced--agrimycin 100, agristrep, phytomycin, acco-streptomycin, etc. They are crude preparations of streptomycin together with terramycin or some other antibiotic. Agrimycin contains 15% streptomycin sulfate and 1.5% oxytetracycline (terramycin) in a powderlike filling. Agristrep is similar to agrimycin but differs from the latter in that it has a higher content of commercial streptomycin (37%). Phytomycin--a liquid preparation, contains 20 % streptomycin and is the most stable upon at storage. Accostreptomycin contains 45% streptomycin is also qulte at stable, and can be preserved for 2 years. Of wide use in plant growing is the antibiotic, actidione.

  Treatment of fruit trees. The best results in the application of antibiotics were obtained in horticulture in the treatment of fruit and nut trees infected with bacteria. Goodman (1954 a, b, c) in Missouri, Young and Winter (1953) in Ohio, Hueberger and Poulos (1953) in Delaware, Ark (1953 a, 1954) and Dunegan (1954) in California, Kienholz (1954) in Oregon, Clayton (1955) in North Caroline, Kirby (1954) in Pensilvania and Mills (1955) in New York obtained good results, upon spraying and dusting antibiotics on plants infected with bacteria. Wherever such treatment was performed the morbidity of apple and pear trees decreased or stopped altogether. According to Goodman, Dunegan, Ark and others, 3-4 sprayings of agrimycin at a concentration of 30-100 µ g/ml completely eliminated the infection of woody plants. Ark sprayed powderlike crude streptomycin and a solution of a purified preparation. In treatment of apple and pear trees afflicted by Bact. amylovorum or of walnut afflicted by Bact. juglandis the pure preparation was superior. In treating nut trees, two sprayings of streptomycin sulfate solution at a concentration of 10 µ g/ml were applied. Dye D. and Dye M. (1954) successfully treated the seedlings of pear trees, infected by Bact. juglandis with a solution of streptomycin sulfate and dihydrostreptomycin at a concentration of 100 µ g/ml.

  Good results are obtained upon treating fruit trees (cherry, etc) infected by Bact. syringae, with actidione. One or two sprayings with a solution containing 1-2 µ g/ml is sufficient to stop the disease. The characteristic spots on leaves cease to appear and those already existing disappear. Today, actidione is used before fruit formation and after the cherries ripened, although investigations show that this antibiotic does not poison the fruits and may be used during fruit bearing (Hamilton and Szkolnik, 1953; Cation, 1953).

  In Germany, Klinkowski and Keller (1956) in their struggle against mildew of fruit trees used crude antibiotics (filtrates of culture fluids) obtained from specially selected actinomycetes. The preparations were applied to the trunks of the apple trees infected with the disease "white ripening", The experiments performed in orchards on a large scale yielded positive results.

  Treating leguminous plants. Mitchel et al., (1952) tested antibiotics--streptomycin, terramycin, neomycin, aureomycin, patulin, subtilin, etc, (in all 12 preparations) in treatment of leguminous plants artificially infected with bacteria. He introduced these bacteria into the plants by the use of a paste, which he smeared on the stems. Under laboratory experimental conditions, kidney beans and soy were totally protected against bacterial wilt by treatment with streptomycin or dihydrostreptomycin. All control plants perished.

  After the successful experiments of Mitchell and co-workers, large-scale field experiments were started for testing antibiotics on leguminous plants. On the experimental fields of Beltsville in Maryland streptomycin was used in the struggle against the fungal disease of Chilean beans, caused by Phytophthora phaseoli. Spraying with an antibiotic solution at a concentration of 100 µ g/ml the morbidity decreased markedly or even disappeared entirely. The application of crude streptomycin in concentrations of 50 µ g/ml gave better results than the use of chemically pure preparations even at higher concentrations. It is assumed that the crude preparation of streptomycin contains some other substance with antifungal properties.

  In fighting mildew of beans, agrimycin at a concentration of 25 µ g/ml in a mixture with copper preparations of the same concentration, was used. This preparation was more effective than the copper preparation and agrimycin applied separately (Zaumeyer and Fisher, 1953; Zaumeyer, 1955).

  Dekker (195 5) tested the action of a culture fluid of the actinomycetes-antagonists--A. rimosus on pea seeds infected with Ascochyta pisi and Mycosphaerella pinodes. The antibiotic substance penetrated the seeds and killed the disease agent therein. Such seeds germinated normally and gave rise to healthy seedlings, while in the control massive infection was observed.

   Klinkowski, Kohler and Shroedter (1955) treated bean seeds with crude antibiotics formed by Penicillium chrysogenum, and A. griseus in order to prevent infection of the sprouts by the bacteria Ps. phaseolicola. The seeds were soaked in antibiotic substances and thus relieved of infection.

  Treatment of vegetable cultures. Brian et al., (1951) treated infected lettuce and tomatoes with griseofulvin. This drug possesses strong antibiotic properties and inhibits numerous species of fungi, including phytopathogenic ones. It does not affect bacteria. Spraying lettuce, infected by the fungus Botrytis cinerea, with a solution of the antibiotic yields quite satisfactory results.

  Similar results were obtained in experiments with tomatoes infected by the fungus Alternaria solani. Griseofulvin was either introduced into the substrate under the root system, or it was sprayed on the leaves. In the control containers the morbidity incidence was 100%, while upon treatment the disease was not apparent at all, or only a small percentage of the plants contracted the disease.

  In one of the experiments the number of spots that appeared as a result of the disease an the leaves of the tomatoes, was counted. In cases where griseofulvin was introduced in a dose of 10- 2 0 µ g/ml per 1 g of substrate there were no spots on the leaves or only very few; in plants not treated with the antibiotic there were more than 1,250 spots on a single plant.

  The antiblotic thiolutin (obtained from A. albus) was used by Gopalkrishmann and Jump (1952) against fusarium wilt of tomatoes (caused by Fusarium oxysporium lycopersici. The authors treated tomato seedlings by dipping their roots in the antibiotic solution before planting them in the soil. This procedure completely protected the plants against the disease. The control plants all succumbed to the disease. Microbiological analysis of the tissues of the plant showed that with small doses (10 µ g/ml) of the antibiotic there were no outward signs of the disease but the mycelium of the fungus could be found Iin the tissues. After treatment with large doses of the antibiotic (40-80 µ g/ml) the tissues of the plants were sterile and no mycelia were observed in them.

  In another series of experiments the authors soaked tomato seeds in the antibiotic and planted them in the soil. After 12 days 100% of the control plants had fusariosis, while the treated plants showed no signs of the disease or only a very small number of them showed its symptoms.

  No less effective results were obtained when antibiotics were used against bacterial infections of tomatoes or other cultures. Conover (1954, 1955) reported good results in treating tomatoes and pepper infected with Bac. vesicatorium, with agrimycin and streptomycin. After 5 sprayings with a solution of the antibiotic at a concentration of 200 µ g/ml, 74% of the plants were completely healthy and only 0.4%, were seriously affected. Among the control plants 12% were healthy and 34% were very sick. Ninety-five per cent of the treated plants were suitable for replanting and among the untreated plants only 27% could be replanted.

  Cox et al., (1953) completely cured diseased pepper by spraying it 3 times with streptomycin at a concentration of 500 µ g/ml. Similar data are given by Crossan and Krupka (1955), who found that upon treatment with the antibiotic the disease agent in the leaves of pepper is totally eradicated. Higher concentrations of the antibiotic, according to Cox (1955), yielded a smaller effect and sometimes even caused an increase in morbidity. The author noted the beneficial effect of a mixture of streptomycin (100-200 µ g/ml) and copper preparations.

  No less effective results are obtained upon treatment of celery infected with Pseudomonas apii, a disease which is very common in Florida. The use of agrimycin (300-600 µ g/ml) almost completely eradicates the disease. As in the case of treating tomatoes and peppers, a mixture of streptomycin and a copper preparation gives better results.

  Sutton and Bell (1954) treated turnips infected by Pseudomonas campestris. They treated the seeds with aureomycin solutions diluted 1:2,500 and 1:1,000 before sowing. Short exposure of the seeds to such a solution completely eradicated the disease agent; the germination of the seeds was normal and even a stimulation of growth of the seedlings was noted. The plants were healthy, while in the control they were infected to an extent of 30-76%.

  The disease of the eyes of potato tubers caused by the bacteria Bact. atrosepticum and Pseudomonas fluorescens is often the cause of serious injury to potatoes in the field and storage. The application of antibiotics in such cases gives very good results. Bonds et al., (1953-1955), at first in greenhouse experiments and later under field conditions, found that treatment of cut tubers of diseased potatoes with a streptomycin sulfate solution (25 µ g/ml) prevents the disease in 80-100% of plants. Dipping of the tubers for a short time in the solution of the preparation not only diminishes the morbidity but also increases the viability of the sprouts. According to Webb (1955), treatment of potato eyes with agrimycin had little effect, however treatment with phytomycin had very good results. The tubers produce more viable plants with abundant flowering and the tuber crop was 15% higher that of the control. Heggested and Clayton (1954) had good treatment results wit tobacco infected with Pseudomonas tabaci. The authors used a streptomycin sulfate solution followed by agrimycin and agristrep in 200 µ g/ml concentrations. These solutions were sprayed on the plants 2-3 times during the summer. As a result, there was almost no plant morbidity, while more than 30% of the control plants perished. The effectiveness of the antibiotic was higher than that of the copper preparations. Beach and Engle (1955) in Pennsylvania achieved excellent results in the treatment of tobacco with antibiotics. They used phytomycin at a 100 µ g/ml concentration. Kirby (1955) treated diseased tobacco plants with agrimycin (100 µ g/ml) mixed with febram. All the authors noted a decrease in morbidity, improvement of growth, increase in number of leaves and also a greater development of the root system.

  Antibiotics were successfully used in treating decorative plants. Robinson, Starkey and Davidson (1954) treated chrysanthemums infected with bacteria with solutions of streptomycin, terramycin, neomycin, chloromycetin and other substances. The best results were obtained with the first three preparations. The antibiotics were introduced into the roots or into the grafts. The treated plants either did not succumb to the disease at all, or the incidence of disease was very low, while the control plants all perished. The antibiotics eradicated the infection in the plant tissues (Pramer, 1955).

  Treatment of grain cultures. Attempts have been made to use antibiotics in diseases of cereals. Wallen (1955) used various antibiotics against wheat rust, caused by Puccinia graminis var. tritici. The rust-effective antibiotic in his experiments was actidione. Concentrations of 50-500 µ g/ml, although toxic to the plants, lowered the morbidity to 0-5 %. At a lower concentration (2 5 µ g/ml) no toxicosis manifested itself and the percentage of morbid plants was within the range of 50-60%, while the morbidity in the control was 100%.

  The crop of the wheat treated with the antibiotic was higher than that of the untreated plots. The percentage of germinating seeds was higher among the experimental plants (more than 90%) than among the controls.

  Leben, Army and Keitt (1953) applied the antibiotic helixin "B" against the disease of oats, caused by Helminthosporium victoriae and against the disease of barley, caused by Helminthosporium sativum. The antibiotic considerably lowered the incidence of disease in the plants. In the control, under conditions of an experiment in growth containers, there was 28-29% of diseased plants and under field conditions--2%; after treatment with the preparation no diseased plant was observed. In the growth containers and under field conditions diseased plants did not exceed 1%. Positive results were also obtained in laboratory experiments, using antibiotics against wheat rust (caused by Tilletia foctens), oat rust (caused by Ustilago avanae), and barley rust (caused by Ustilago hordei).

  Henry et al., ( 1952 -1953) achieved an almost complete eradication of the rust disease of wheat by treatment with actidione mixed with a preparation called "Dixie clay." Even better results were obtained when actidione was used as a dust mixed with "Dixie clay" and a preparation called "Captan", which alone did not give good results in the struggle against the mentioned diseases.

  Antibiotics have not so far been effective in combating virus diseases of plants. Attempts were made to use various antibiotic substances against tobacco mosaic (Schlegel. David and Rawlins (1954) and certain other virus diseases (Leben and Fulton, 1952); the small positive effect sometimes observed was not caused by suppression of the virus particles but by the action of the antibiotics on the host plant, which increased its growth and enhanced the resistance of the tissues (Zaumeyer, 1955).

  The given data on the use of antibiotics in plant growing are as yet scanty. However, there is a basis for hope that these substances will prove to be no less effective in the treatment of plants than in the treatment of animals and human beings. Experiments show that a number of antibiotics may already be widely applied in agriculture, in the struggle against fungal and bacterial diseases of woody and grassy plants.

  Antibiotics are in many cases not inferior in their action to modern antiseptics, and often surpass them. It is possible that in the future, with the study of conditions and the mechanism of action, and with improvement of the methods of their application, antibiotics will become even more effective.

  If the antibiotics, after their introduction into the plant stems are directed into the root system, and accumulate there at a higher or lower concentration, then these preparations would probably be effective against root diseases; this might be of great importance.

  One also should not overlook the economic side of this enterprise.

  At first one should assume that it will be advisable to apply antibiotics to the most costly cultures, mainly in horticulture, in the struggle against diseases of fruit and decorative plants.

  In these cases, not only the cost of a given treatment of the tree is of importance, but also the time necessary for growing a fruit-bearing plant, should be considered. Antibiotics are less harmful to the health of man than antiseptics. A certain portion of the chemical substances which enter the plant tissues, when they are treated with antiseptics, concentrate there, and to some degree lower the nutrient qualities of the plants both as food and fodder. The possibility is not excluded that certain elements may prove to be harmful to man and animals.

  Antibiotics are less dangerous in this respect. They cannot accumulate in concentrations which are toxic to animals and man. One may choose antibiotics which are altogether harmless. Certain antibiotics have an activating effect on plants and increase their growth. All this illustrates the necessity of devoting more attention to the antibiotics than has been done until the present.

 





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