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PART IV, continued:

 

Microbial Antagonists

  Among the soil microorganisms there are forms thatinhibit the growth of other microbes. They are usually called antagonists.

  There is no fundamental difference between inhibitorsand antagonists. Both groups act with the aid of special metabolites in their metabolicproducts: the inhibitors affect cells of higher organisms and the antagonists acton lower organisms. Even this distinction is not always sharp, since there are inhibitors,which also suppress microbes and, on the other hand, there are antagonists that arealso toxic to plants.

  As was already mentioned, substances formed by inhibitorsare called toxins or phytotoxins, and substances produced by antagonists are calledantibiotics. This differentiation is of a purely formal or conventional nature. Asis well known, there are many substances among the antibiotics which are highly toxicto plants and animals.

  Regardless of this relativity of concepts and designationsthe antibiotics and their producers are a special branch of science and are consideredas special substances with specific manifestations.

  Microbial antagonism has caught the attention of scientistsfor many years. Pasteur, Mechnikov and their contemporaries noticed the ability ofsome species of microbes to suppress the growth of others (cf. Nakhimovskaya, 1937;Waksman, 1947; Krasil'nikov, 1950 and others). Pasteur observed this ability in theanthrax bacillus in relation to the microbe causing chicken cholera. Mechnikov foundit in the lactobacilli in relation to the putrefactive bacteria and certain colon-typebacilli. On this basis he devised a method of changing the intestinal flora and sanitationof the human and animal intestines. The phenomenon of antagonism was observed invarious groups of microorganisms, among bacteria, fungi, actinomycetes, algae, protozoa,etc. Microbial antagonists acting upon various pathogenic bacteria against cocci(staphylococci, streptococci, pneumococci and diplococci), against organisms causingintestinal infections (dysentery, parathyphoid, typhoid and cholera) against thetubercle bacillus, diphtheria, peat and anthrax brucellosis, tularemia and gas gangrenehave been described. A great number of antagonists were described acting againstpathogenic fungi, yeasts, protozoa, etc.

  Recently, the efforts of investigation have been concentratedon the detection of antagonists acting against viruses and malignant tumors, Antagonistsof viruses and tumors can be found among actinomycetes and bacteria (Waksman, 1953;Kashkin, 1952; Kurylowicz and Slopek, 1955).

  Much data are available on the suppressing action ofantagonists of phytopathogenic bacteria, actinomycetes and fungi. Antagonists werefound acting against various organisms. The greatest attention is paid to antagonists--actinomycetes.

  Among 20 cultures of actinomycetes isolated from rhizospheresoil, Lochhead and Lauderkin (1949) found 11 cultures that suppressed the growthof phytophathogenic strains of Actinomyces scabies.

  Meredith and Semeniuk (1945-47) found that among theactinomycetes which they studied 21% inhibited growth of the fungus Pythium graminicola,which causes root necrosis in a number of plants. The actinomycete-antagonists ofChalaria quercina which cause wilt in the oak, and actinomycete-antagonistsof Cerasto mella ulni, which affect woody plants, such as the elm, etc havebeen described (Stallings, 1954).

  From the soils of the southern shore of Crimea, Petrusheva(1953) isolated 31 cultures of actinomycetes. Twenty-two of these inhibited the growthof the fungi Thielaviopsis basicola which causes root rot of tobacco plants,and Fusarium sp. which causes the "black foot" disease of citrussaplings, Leben and Keitt ( 1948) had a collection of actinomycetes that inhibited33 species of phytopathogenic fungi.

  Cooper and Chilton (1950) have done much work on thedetection of actinomycete-antagonists of the phytopathogenic fungus Pythium arrhenomonaswhich is widely distributed in the soils of Louisiana. Out of 8,302 strains whichthey isolated, 18.5 to 31.5% were antagonists. Kublanovskaya (1950) noted actinomycete-antagonists to the agent of wilt of the cotton plant-- Verticillium dahliaeand Fusarium vasinfectum. Lechevalier et al., (19 5 3) tested 197 strainsof actinomycetes for antagonism to Cerastomella ulni and found only one activestrain. Actinomycete-antagonists were found in soils which were active against phytopathogenicfungi--Helminthosporium sativum, H. victoriae, Coletotricum circinans, Verticilliumalbo-atrum and others (Stevenson, 1954; Stessel et al., 1953).

  In our studies we found actinomycetes in soil, whichsuppress phytopathogenic fungi., Fusarium lin, F. solani, F. vasinfectum, Helinthosporiumsativum, Alternaria humicola, Rhizoctonia solani, Botrytis alii, Deuterophoma tracheiphilus,Trichoderma lignorum, Monila fructigena, and also fungi of the genera Penicillium,Aspergillus, Cladosporium, Verticillium and others.

  Among fungi there are many antagonists. Antagonistshave been described against agents of various diseases: Fusarium, Peziza, Rhizoctonia,Ophiobolus, Botrytis, Monilia, Sporotrichum, Pythium, Phymatotricum, Phytophthoraand Sclerotium.

  Porter (1924) described antagonistic relations betweenfungi and bacterial antagonists, and the phytopathogenic fungi Helminthosporiumand Fusarium. Sanford and Broadfoot (1931) isolated from the soil 6 speciesof fungi which inhibit the growth and activity of the fungus Ophiobolus graminis.Weindling (1932, 1948) described a case of parasitism of the fungus Trichodermalignorum on the fungi of the genus Rhizoctonia and others. The same wasobserved by Novogrudsk (1936). The latter given a long list of fungi and bacterialantagonists, indicating the species of fungi and bacteria inhibited by them.

  In this list appear more than 30 fungal species whichinhibit over 50 fungi belonging to different genera and families. Many other investigatorsalso found antagonists among the fungi (Allen and Haenseler, 1935; Joseph, 1952;Vasudeva, 1950, 1953 and others).

  Stemmel, Leben and Keitt (1953) isolated 170 fungiantagonistic to other fungi from soils.

  Anwar (1949) found that among soil fungi and bacteriaapproximately half (out of 86 studied) suppressed the growth of the fungus Helminthosporiumsativum, and about 12% inhibited Fusarium lini. From various soils, Gregoryat al., (1952) isolated 14 cultures of fungi, 29 strains of actinomycetes and 31strains of bacteria, which actively suppressed the growth of the fungus Pythiumdebaryanum. Three actinomycete strains and 1 bacterial strain suppressed growthof the root-nodule bacteria, Rh. meliloti and Rh. trifolii.

  Among the sporeforming and nonsporeforming bacteria,it is not uncommon to encounter antagonists to phytopathogenic microbes. Among thesporeformers antagonists to various phytopathogenic fungi have been described. Porter(1924) mentions a number of bacteria which inhibit the growth of Helminthosporium.The latter did not grow in the presence of Bac. capoulatus, Bac. mesentericusand Bac. mycoides. Bamberg (1931) indicated inhibition of growth of the fungiTilletia tritici, Ustilago zeae, U. levis, U. avenae by the sporeforming bacillusBacillus D.

  The afore-mentioned bacteria showed similar actionagainst phytopathogenic fungi such as Penicillium sp., Helminthosporium,Ophiobolus, Acrostalagmus, Fusarium. Sclerotinia gleosporium, Alternaria andothers (Novogrudskii, 1936; Weindling, 1946; Stessel et al., 1953; Stallings, 1954;Krasil'nikov, 1953 a and others).

  Many antagonists have been described among the nonsporeformingbacteria. Studies show that they are most frequently encountered among the Pseudomonasand Bacterium species and also among myxobacteria. Bisby (1919) describedthe species Pseudomonas phaseoli which inhibited the fungus Fusarium oxysporium.Fawcett (1931) indicated Ps. juglandis as an antagonist of the fungus Dothlorellagregaris. Johnson and Marvin (1931) detected the antagonistic action of certainnonsporeforming bacteria (Bacterium C-1. Bacterium X, etc) on growthof the fungi Ustilago zeae, U. avenae, Alternaria solani, A. brassicae andA. Tenuis.

  Khudiyakov (1935) isolated and studied in detail bacteriawhich dissolve the mycelium of the fungi Fusarium graminearum, F. culmorum, F.scirpi, F. lini. F. herbarum, F. equiseti, Sclerotinia libertiana and others.These bacteria were called mycolytic bacteria. Subsequently, many other investigatorsdetected these bacteria in the soil ( Raznitsyna, 1942; Berezova, 1932; Korenyako,1939; Kublanovskaya, 1953, etc).

  Ark and Hunt (1941) had cultures of the bacteria Bac.vulgatus and Bac. sp. that inhibited the growth of many phytopathogenicbacteria--Bact. amylovorum Bact. aroideae, Bact. carotovorum, Bact. phytophthorum,Ps. campestris, Ps. lachrymans and others. These bacteria also inhibited certainfungi--Fusarium graminearum, F. lycopersici, Phytophthora sp. and others.Similar data are given by other authors (Johnson, 1935, Weindling, 1946; Christensenand, Davis, 1940; Vasudea, 1952 and Skinner, 1956).

  Antagonists to phytopathogenic fungi have been describedamong the myxobacteria (Johnson and Marvin, 1931; Kononenko, 1937).

  Fungi which attack nematodes are described in the literature.They were first described by M. S. Voronin in his work "Mycological Studies"published in 1869 and by Sorokin in 1871. In a series of papers Soprunov (1954) showedthat these fungi differ in their species composition and are very widespread in soils.The majority of them belong to the Hyphomycete, to the genera Trichothecium,Arthrobotrys, Dactylaria, Dactylella, etc.

  These fungi "catch" the nematode with theirhyphae and poison it with their metabolites. Attempts were made to use these fungiin the struggle against phytopathogenic nematodes. The introduction of this fungusinto the soil lowers the incidence of plant disease. In the struggle with nematodeswhich affect cucumbers, the fungi-antagonists, or an they are called the predatoryfungi noticeably decrease the incidence of disease; in the control group there were23 galls per each plant and in the treated plants, an average of 0.6 galls (Soprunov,1954).

  According to Gorlenko (1955), artificial enrichmentof soil by predatory fungi lowers the morbidity of cucumbers 1.5-7 times. Tendetnik(1957) used the predatory fungi for exterminating pathogenic larvae, the ancylostomesin mines and also to destroy strongyles in the manure of infected animals.

  On the basis of existing evidence, one may say thatthere are no species of bacteria. actinomycetes, proactimomycetes, micromonospores,protozoa, algae, etc against which no antagonist can be found. In laboratory practice,one usually gives the name antagonist only to a microbe which suppresses the widelyused test organisms, which usually comprise a narrow range of known cultures of bacteriaor fungi. One does not take in account the fact that the so-called inactive forms(nonantagonists) in these tests might be active against other organisms. On the basisof our own experience and data from literature, we can say that the property of antagonismis characteristic of all species of microorganisms, but is expressed differentlyand to various degrees depending on the natural properties of the antagonist on theone hand, and the sensitivity of the test organism on the other hand, and also uponthe type of substrate and other external conditions.

  The antimicrobial action of the antagonists manifestoitself not only under laboratory conditions on artificial media but also under naturalconditions of habitation, in the soil. In sterile soil, where there are no antagoniststhe growth of microbes and the biochemical processes which they cause take placeat an intense rate. However, it is sufficient, to introduce an antagonist in suchsoil in order to stop or to slow down the growth and biochemical processes of microbe.

  In his experiments, Afrikyan (1951) directed the actionof antagonists of the group of sporeforming bacteria: Bac. subtilis and Bac.mesenterics against the organisms: Bac. mycoides and Az. chroococcum.The result are given In Table 110.


Table 110
Intensity of Azotobacter growth as depending on
growth of the antagonistic bacterium--Bac. mesentericus
(experiment with sterile soil; table shows number of cells, in thousands,
in 1 g of soil after passage of days)

Experimental conditions

Initial number

1

2

3

5

7

10

No antagonist (control)

80

150

380

520

760

800

1,000

With antagonist

80

100

30

0.05

0

0

0.05

No antagnist (control)

200

500

600

800

800

800

500

With antagonist

200

100

50

0.5

0

0

0.05

Antagonist

80

400

1,200

1,500

1,600

1,000

800

  Bac. mesentericus was introduced into sterilizedsoil together with cultures of Azotobacter to which it is an antagonist, Thegrowth of Azotobacter was suppressed by Bac. mesentericus. Under conditionsof growth in sterilized soil when grown alone, Azotobacter reached 1 millioncells per 1 gram of soil and in the presence of Bac mesentericus, Azotobactergrew very slowly and only during the first day; then the number of its cells decreasedrapidly, dropping practically to zero. Only at the end of the experiment, after 10-15days, when growth of the antagonist and formation of the antibiotic stopped, didthe Azotobacter resume growth although at a very slow rate.

  The same was observed in experiments with Bac. mycoides.This microbe in very sensitive to the antibiotic action of the potato bacillus. Inan isolated condition, in sterilized soil, its growth is excellent but it ceasesto grow completely or almost completely in a mixture with the antagonist (Figure92).

 

Figure 92 Growth of Bac. mycoides in soil (sterile) in presence of the antagonist--Bac. mesentericus:

1--growth of Bac. mycoides without antagonist; 2--growth of Bac. mesentericus in soil; 3--growth of Bac. mycoides in a mixture with Bac. mesentericus.

 

  Mikhaleva (1951) studied the growth of the root-nodulebacteria of clover, peas, kidney, beans, lupine, soy, lucerne, etc in the presenceof antagonists and actinomycetes, The most resistant, according to their data, werethe soy bacteria In podsol soils these bacteria are more strongly suppressed by actinomycetesthan in chernozem soils. Root-nodule bacteria of clover perish after 4 days in thesoil in the presence of the antagonist.

  The activity of the root-nodule bacteria decreasesmarkedly under the influence of the antagonist; the number of nodules on the rootsis usually smaller than in the controls. According to our observations, the root-nodulebacteria of clover vetch and peas react noticeably to the action of antagonisticBac. subtilis, Bac. mesentericus and others. When the soil is artificiallyenriched with these bacteria the nodules do not develop on the roots of the above-mentionedplants or they appear in small numbers only, with a degenerated appearance, an unusualform and a small size.

  Stolp (1952) studied the growth and activity of root-nodulebacteria of peas in the presence of Pennicillium expansum. Robinson (1946)observed cultures of antagonists among actinomycetes, bacteria and fungi, which activelysuppressed the growth and virulence of root-nodule bacteria in the laboratory andunder field conditions as well.

  The general importance of bacterial antagonists isdetermined not only by the nature and strength of their activity but also by theirnumber in the soil. The more intensely they grow in the soil, the higher their concentrationand the stronger the effect they exert.

  It is difficult or almost impossible to assess thetotal number of antagonists in the soil. As was already mentioned, all microbes possessantagonistic proprties against some microbe. However, it is practically impossibleto detect antagonism against all existing microorganisms.

  In our studies we give quantitative indexes of distribution.of antagonists in relation to certain species of bacteria or fungi. Among the bacteria,cultures of Azotobacter, root-nodule bacteria, mycobacteria, micrococci andnonsporeforming bacteria (colon bacillus, etc) were used.

  According to our calculations, there are very largequantities of actinomycete-antagonists with clearly expressed antimicrobial propertiesin different soils.

  In the previous chapter (microbial inhibitors) datawere presented on the number of microbes which suppress the growth of Azotobacter.The number of bacterial antagonists ranged between 10,000 and 450,000; fungi, between1,300 and 17,000 and actinomycetes, from 10,000 to 160,000 in 1 g of soil, dependingon the properties of the latter.

  In relation to certain other species of bacteria(Bac. mycoides, Bac. subtilis) and fungi (Fusarium lini, Fusarium sp.,etc) the total number of antagonists is much higher. In our analyses of various soilsof the Soviet Union we found tens and hundreds of thousands and often millions ofthem in 1 g of soil.

  In the podsol soils of the Moscow area one may find40,000- 1,000,000 actinomycetes-antagonists or even more, per 1 g of soil; sporeformingbacteria in numbers of 20,000-500,000 may be found in 1 g of soil. In the chernozemsof the southern districts of the USSR the total number of microbes is higher and,as a rule, the number of antagonists is also higher. However the percentage of antagonistsmay be lower. For example, the soils of the Crimean steppes contain a smaller percentageof antagonists than the northern soils of the Kola Peninsula or the soils of thecentral districts. There are relatively few antagonists in the red soils of the Caucasiancoastal area (Table 111).

Table 111
Number of actinomycetes-antagonists in soils (in thousand/1 g soil)

Soil and region

Actinomycetes

Antagonists

Serozem, Tashkent

560

200

Serozem, Vakhah valley

1,300

360

Chernozem, Kuban'

2,000

400

Chernozem, Kar'kov

1,000

400

Chernozem, Kuibyshev

1,500

800

Chernozem, Crimean steppe

1,200

120

Krasnozem, Batumi

200

10

Chestnut soil, Armenia

600

200

Brown soil, Armenia

1,200

600

Chernozem, Armenia

800

200

Chernozem, Georgia

1,800

350

Podsol, Moscow, Oak forest

1,200

350

Podsol, Moscow, Spruce forest

360

0

Podsol, Moscow, Birch forest

1,200

420

Podsol, Moscow, tilled, a

1,500

800

Podsol, Moscow, tilled, b

600

200

Podsol, Leningrad, tilled

1,500

800

Podsol, Kola Peninsula, ferruginous

0.4

0.3

Humus, Kola Peninsula, ferruginous

2.0

1.5

Tundra, Kola Peninsula, forest

0

0

Swampy soil, Kola Peninsula

0.2

0.2

  In certain soils one may find 2,000-400,000 bacterialantagonists per 1 g of soil; they belong to two groups--Bac. subtilis andBac. mesentericus. Among the nonsporeforming bacteria and especially amongspecies of the genera Pseudomonas and Bacterium, there are many antagonists.A special place in relation to their quantity among the antagonists of this groupis occupied by the mycolytic bacteria. These bacteria grow well in many soils andin the rhizosphere of various plants. According to our figures, their total numberapproaches tens of millions and more in 1 g of soil (Krasil'nikov, 1940 a,b, c; Kusina,1951; Kublanovskaya, 1953).

  Rasnitsyna (1947) studied the soils of the Vakhah valleyand counted 100,000 to 100 million mycolytic bacteria (which lyse the fungus Fusariumvasinfectum) in 1 g of soil, depending upon the vegetative cover. The greatestnumber was found in the rhisosphere of lucerne, spear grass and the Euagropyrum;they were less numerous under rye grass, brome grass and peas. Kuzina (1955) givesmore or less similar indexes for light serozems of the Uzbek SSR.

  Novogrudskii (1949 a) isolated mycolytic bacteria fromKazakhstan soils which lyse the fungi: Fusarium culmorum, F. graminiarum, Verticillumdahliae, Colietotrichum lini, Alternaria tenuis, Amblyosporium botrytis, Pyronemaconfluens, and Mucor racemosus.

  In the same soils the author counted from 100 to 100thousand mycolytic bacteria in 1 g of soil. These are active solely against Fusariumgraminiarum and were more numerous under lucerne than under oat or millet.

  Above were given data on growth and accumulation ofthese bacteria in soils under different plants.

  In nature microorganisms do not live alone, but inassociations which contain many foreign species--competitors and noncompetitors.In these associations definite and quite complicated relations are established amongthe species, of both a symbiotic and an antagonistic nature.

  In microbial populations or coenoses, each speciesin its struggle for survival throughout a long history of evolution, elaborated certainmeans of struggle with its competition. These means are versatile. Microbes may displacetheir competitors by abundant multiplication or they may form during their metabolismvarious specific and nonspecific substances which suppress microbial growth. Thesenonspecific substances are, for example, organic acids, alcohols, peroxides and othercompounds. These metabolic products are characteristic of many microbial species.

  Examples of the nonspecific metabolites are certaininorganic substances, such as ammonia, hydrogen sulfide, ions of chlorine, SO3,aluminum, iron and other elements, Plentiful emission of H2S, formed asa result of the vigorous metabolic activity of the appropriate microbes, may causethe poisoning of the milieu, making it inadequate for the growth of many foreignspecies of microbes and even for higher plants, as was observed by us in soils withhigh ground-water level in the Trans-Volga region (Krasil'nikov, Rybalkina and others,1934).

  Microbes producing acids, alcohols and other organiccompounds suppress the growth of organisms which are sensitive to these substances,regardless of the species to which they belong.

  The most characteristic and outstanding reactions arethose which are caused by particular specific substances, the so-called antibiotics,which act against microbes. These substances have specific effects. The microbialantagonists which form these substances suppress growth of certain specific speciesonly. Some antagonists inhibit only gram-positive bacteria and others, inhibit bothgrarn-positive and gram-negative bacteria. Some of them act on cocci and others onbacilli.

  Some antagonists have an inhibitory effect only onfungi or only on phages and viruses, etc.

  Antibiotic substances produced by antagonists are apotent weapon in the struggle with competing microbes.

  A characteristic feature of such antagonists is thefact that as a rule, they act only upon foreign species. A. streptomycini,the producer of streptomycin, does not inhibit cultures of its own species. The producerof aureomycin, A. aureofaciens does not inhibit cultures of its own species,regardless of from where they were isolated or under what conditions they lived previously.Similarly, other antagonists which produce antibiotic substances, terramycin, chloromycetin,actinomycin, sulfactin, etc do not suppress the growth and development of strainswhich belong to the same species.

  These characteristics of specific or selective actionof antagonists determine to a large extent the species makeup of microbial populationsin natural substrates.

  Experience shows, that there are microorganisms thatform antibiotics under conditions of solitary growth, on artificial nutrient media.Their ability to form these antibiotic substances is hereditarily fixed and expressesitself in the absence of competitors.

  These organisms are looked upon as potent antagonists.They are often found among various microbial species. They comprise the main groupof producers of antibiotic substances obtained to date in various laboratories andin the antibiotic industry. In other species this property is not fixed by heredityand appears only in the presence of competitors, i.e., under conditions of mixedpopulations. In pure isolated cultures these organisms do not produce antibioticsubstances. For example, the colon bacillus, Bact. coli suppresses the growthof Bac. anthracis only in those cases where both organisms are together. Ina pure culture the colon bacillus does not produce antibiotic substances which areactive against the mentioned microbe. Apparently these microbes only produce antibioticsubstances under pressure, out of necessity.

  Shiller in 1914 was the first to pay attention to theforced nature of the formation of antibiotics by microorganisms. He was working inMechnikov's laboratory on the interrelations between the acidophilic bacilli, sporeformingbacteria, streptococci, pneumococci and other species (Shiller, 1952).

  The phenomenon of forced antagonism was also noticedby other investigators (Peretts and Slavskaya, 1934; Izabelinskii and Soboleva, 1934and others). Streshinskii (1949, 1950) has demonstrated the formation of antibioticsubstances in mixed cultures of a fungus and a sporeforming bacillus. According tohis observations, Bac. subtilis forms an antibiotic substance against thePenicillium fungus only when the two grow together. The fungus too becomesa more active antagonist in the presence of the bacillus. We observed forced antagonismin a number of actinomycetes. Cultures grown separately on nutrient media, do notform antibiotic substances, but in the presence of certain microbes (fungi or bacteria)these substances which suppress the growth of competitors are formed. Two inactivespecies of actinomycetes when grown together, form antimicrobial substances.

  All such organisms possess a latent antagonistic capacitywhich is not fixed hereditarily.

  The majority of microbial antagonists readily losetheir ability to produce antibiotics, active strains turning inactive in the processof variation. This property is encountered in many organisms used in industry andcauses many difficulties in the antibiotic industry and in laboratory practice.

  Antibiotic substances should be classified an potentweapons in the struggle of microbes with neighboring competitors; as a biologicallyimportant attribute formed in complex populations during phylogenesis and as a propertydetermining the degree of development and distribution of the microorganism in nature.The biological role of antibiotic substances and, therefore, the phenomenon of antagonismas a whole, is quite important in the life of both higher and lower soil organisms.

  Through use of their metabolic products antagonisticmicrobes suppress their competitors, removing them from the substrate and thus exertinga definite selective action. To a certain degree, microbial antagonists regulatethe formation of microbial coenoses (colonies) in the soil in general. They playan important role in the improvement of soils, in the so-called process of self-purificationof soils. The removal of harmful pathogenic and phytopathogenic flora and fauna isaccomplished by microbial antagonists.

  Microbial antagonists suppress not only the growthand propagation of competing organisms, but also many of the functions of the latter.There are among microorganisms those which inhibit certain processes which occurin microbes.

  Inhibitors were observed to suppress nitrification,denitrification, nitrogen fixation, decomposition of cellulose, etc. Appropriatecultures inhibit the decomposition of organic substances, fermentation of sugars,etc. Microbial cultures are known which inhibit the formation of certain metabolites,various acids, enzyme biotic substances, auxins, vitamins, amino acids and otherbiocatalysts. There are organisms in the soil which, with their metabolic products,suppress the formation of toxins and antibiotics and often inactivate them in themedium, if they are formed there.

  There are data in the literature dealing with the suppressionby inhibitors and their metabolites of processes of cell multiplication, spore formation,budding and the sexual process. Inhibitors often inhibit the process of respiration.

  In the presence of antagonists or their metabolic productsmany active substances: biocatalysts, antibiotics, toxins, and enzymes lose theirpeculiar functions, and become inactive. Toxins become harmless, antibiotics no longersuppress the corresponding microbes, enzymes no longer cause the decomposition oforganic matter, etc. In general, one may say that for any metabolite, living naturecreates an antimetabolite (Woolley, 1954). The metabolite penicillin becomes nonbactericidalfor staphylococci and certain other bacteria, which produce an antimetabolite penicillinase(Abraham and Chain, 1940; Woodruff and Foster, 1945). Sulfonamide compounds are inactivatedby bacteria which produce paraaminobenzoic acid (Woods, 1940; Sevag and Green, 1944,1950) or the factors H.P. etc (Moller and Schuerz. 1941; Green 1940). Mucin suppressesthe antibacterial action of tyrothricin; tannic acid neutralizes actinomycin (Waksman,1947). In our bacterial collection there were strains, which completely inactivatedthe antibiotic substances, formed by the sporeforming bacteria: Bac. subtilis,Bac. mesentericus and Bac. cereus. We also possessed bacterial cultures,which annuled the toxic effect of Ps. pyocyanea metabolites. The toxin ofthe fungus Botrytis cinerea is neutralized by metabolites of certain actinomycetes.

  A solution of mycetin at a 1:10,000 dilution completelysuppresses the growth of Staph. aureus and in the presence of filtrate orcells of a Bac. proteus culture the antibacterial effect is either nonexistant,or becomes very weak. A culture of the colon bacillus and certain Pseudomonasstrains had such an inactivating effect in our experiments. An inactivating effectof microbes was also found in relation to other antibiotics (Krasil'nikov and Nikitina,1951).

  As seen from the above-mentioned data, antagonistsexert a definite suppressing effect on various microorganisms. Due to their action,they have a considerable influence on the formation of microbial coenoses in soilin general and determine, to a certain degree, the distribution and accumulationof the various species of soil microflora.

  Antagonists, therefore, can be considered as one ofthe powerful factors governing soil fertility and plant-crop abundance.

  It is only natural that this group of microorganismsattracts the attention of specialists in many fields--microbiologists, phytopathologists,plant breeders and others. The possibility of practical utilization of antagonistsis one of the most important reasons for the study of the phenomenon of antagonism.

 

The protective role of microbial antagonists

  As was noted before, in soils in which antagonistsgrow abundantly (bacteria, fungi or actinomycetes). microbes, sensitive to them,saprophytes as well as phytopathogens, grow much more slowly, or not at all. Thisserved as a basis for the use of microbial antagonists in the struggle against harmfulmicroflora, and against organisms causing plant disease.

  The first attempts in this direction were made by Porter(1924). He treated wheat seeds with bacterial antagonists and then infected themwith Helminthosporium fungi. The seeds either did not become infected andgerminated normally, or they were slightly affected. Bamberg (1931) infected wheatseeds in order to protect them against smut. Weindling (1940, 1948) used the fungusTrichoderma lignorum for the protection of citrus saplings from Rhizoctonia.This fungus according to other authors, also protects cucumbers and peas from Rhizoctoniaand wheat from fusariosis (Allen and Haenseler, 1935; Bisbu, James and Timonin, 1933).Milliard and Taylor (1927) observed a protective effect of the actinomycetes-antagonist---A.praecox in relation to the agent of scab in potatoes A. scabies. No scabwas observed upon prolific growth of the antagonist.

  A similar phenomenon was described by Kissling (1933).He experimented with bacterial antagonists in subduing potato scab. Under conditionsof a field experiment, upon sufficient growth of the antagonist the disease eitherdid not appear at all or had a limited occurrence only.

  Gregory, Allen, Riker and Peterson (1952) demonstratedthe protective role of actinomycete-antagonists in their experiments with the phytopathogenicfungi Pythium ultimum and Pythium debaryanum. These fungi were quitevirulent for lucerne. The introduction of certain species of actinomycotes-antagonistsinto the soil (Actinomyces sp.) protected the plants from the disease. A positiveresult was also obtained through use of antagonists--fungi Trichoderma lignorumand Penicillium patulum, and the sporeforming bacteria, Bacillus sp.No 6.

  Rehm (1953) treated wheat and rye seeds with actinomycetes-antagonistsin order to fight the organisms which cause fusarlosis (Fusarium nivale andFus. culmorum). As a result, seed germination Increased by 30 %.

  Gorrard and Lockhead (1938) compiled a long list ofmicrobial antagonists which suppress the development of phytopathogenic organismsin soil. Among them there are sporeforming and nonsporeforming bacteria, fungi, actinomycetesand protozoa. Cordon and Haenseler (1939) have shown experimentally the inhibitoryeffect of the sporeforming Bacillus simplex on the growth and developmentof the fungus Rhizoctonia solani which affects cucumbers and peas. A cultureof the antagonist was introduced Into the soil in which the plants were grown. Asa result, the morbidity of the latter dropped. In the control without bacterial antagonists65 % of the total number of cucumber plants and 48% of peas were affected; afterintroduction of the antagonist the mortality role was 35 and 45% respectively.

  Ark and Hunt (1941) mention two cultures of sporeformingbacteria--Bac. vulgatus and Bacillus sp. which suppressed the growthof many phytopathogenic bacteria in soil and protected the plants from disease. Otherinvestigators also speak of the antagonistic action of microbes in soil against phytopathogenicbacteria and fungi (Johnson, 1935; Eaton and Rigler, 1946; Allen and Haenseler, 1935;Feeney and Garibaldi, 1948; Kenknight, 1941 and others).

  In the Soviet Union, as already mentioned above, Khudyakov(1935) and Novogrudskii (1936) established the lytic effect of mycolytic bacteriaon phytophatogenic fungi. These bacteria were subsequently exclusively tested fightingplant infections under laboratory conditions, and in open fields as well. The resultswere positive in many cases.

  The percentage of mortality noticeably decreased. Forexample, the experiments of Berezova (1939), performed on kolkhoz fields with flax,have shown, that mycolytic bacteria lower the incidence of fusariosis by an averageof 40% and in isolated cases their action was even more effective.

  Raznitsyna (1942) used mycolytic bacteria which areisolated from the soil against fusariosis of pine saplings (Figure 93). Under open-fieldconditions on a sector strongly affected by fusariosis, inoculation of bacteria gavean outright positive effect, and the lowering of morbidity reached 80 % and more(Table 112). The plants looked considerably healthier on sectors treated with mycolyticbacteria, than in the control. The needles were longer, stronger and greener, thestem of the saplings thicker and taller than in saplings not treated with bacteria(Figure 94).

 

Figure 93. Protective action of bacteria in fusariosis of pine saplings:

1--plants infected with the fungus Fusarium; 2--plants also infected with Fusarium, but treated with mycolytic bacteria.

 

Table 112
Protective action of bacterial antagonists in fusariosis of pine saplings sown in May 1940

Experimental conditions

Seeds germinated per 100 planted

Number that survived until September

Height of plants in September, cm

Control (not inoculated with bacteria

40

5

3.0

Inoculated with bacterial culture No. 30

75

61

4.9

Inoculated with bacterial culture No. 77

65

46

3.9

Inoculated with Euagroypyrum compost

70

46

4.1

 

Figure 94. Pine saplings growing on sectors affected by fusariosis:

a--not treated with bacteria; b and c--treated with mycolytic bacteria.

 

  Korenyako (1940) studied mycolytic bacteria in thesoils of the Uzbek SSR for a number of years. She isolated cultures of bacteria ofthe genus Pseudomonas, which showed antagonistic properties against the agentscausing wilt of the cotton plant Verticillium dahliae. When these bacteriawere tested on the experimental fields of STAZRA SOYUZNIKHI (Tashkent) which werestrongly affected by the mentioned fungus, positive results were achieved. The mycolyticbacteria suppressed the growth of the fungus and lowered the morbidity of the cottonplant by 60- 80%. Kuzina (1951) and Kublanovskaya (1953) corroborated these results.They showed that mycolytic bacteria in mixtures with other bacteria suppress phytopathogenicfungi more vigorously.

  Davydov (1951 used mycolytic bacteria against the mildeworganism (Sphaerotheca mors uvae) on the gooseberry shrub. The parasitic fungusdisappeared upon introduction of the bacterial antagonists and the plant recoveredand developed normally.

  Petrusheva (1953) used cultures of actinomycetes asantagonists against tobacco-seedling rot caused by the fungus Thielaviopsis basicola,and against blackleg of citrus cultures, caused by Fusarium sp. In soil treatedwith antagonists the plants grew normally; when the actinomycetes were not introducedinto the soil, the mortality of the plants reached 70%.

  Gurinovich (1953) used cultures of actinomycetes andbacterial antagonists in the struggle against the vascular disease of cabbage causedby nonsporeforming bacteria Pseudomonas campestris. The antagonists were introducedinto the soil affected by the soil microbe, and prevented the plants from becominginfected.

  Seiketov (1951) tested four cultures of the fungusTrichoderma--T. glaucum T. lignorum, T. koningii and T. album as antagonistsof Rhizoctonia, which affects potatoes. The percentage of mortality of the"early rose" variety was considerably lower (7-8%) in the experimentalplants than in the controls (54 %).

  Kuzina (1955) used mycolytic bacteria against wiltof the cotton plant caused by verticillium. She treated the seeds with bacteria beforesowing. The morbidity in the control sectors was 54% and after treatment with thebacterial antagonists it was 8--9%. Correspondingly, the crop yield was: in the control-1, 030 bolls and the total weight of seed cotton was 342 g. In the experimental plantsthere were 1,630 bolls and the weight of the seed cotton was 514 g, i.e., 57% higherthan those of the control.

  Kublanovskaya (1953) used actinomycetes as antagoniststo fight cotton-plant wilt. She prepared a special composted preparation of cottoncake with actinomycetes. This was introduced into the soil in which the cotton seedswere sown. The actinomycetes introduced in this manner proliferated abundantly inthe soil and suppressed the growth and activity of the fungi Verticillium dahliaeand Fusarium vasinfectum, protecting the plants from the disease. At the endof the growth period the number of plants in the cotton plants of the 8517 varietyaffected by the wilt was: 52.6% of all control plants and 18.1% in sections treatedwith actinomycetes; in the cotton plants of the 108-F variety there were 23.3 % diseasedcontrol plants and 3.3 % treated plants. In the treated sections all the plants lookedstronger, the bushes were larger, the leaves wider and flowering was more abundant,etc (Figure 95).

 

Figure 95. Protective effect of actinomycetes-antagonists against cotton-plant wilt caused by Verticillium:

a) plants affected by wilt, not treated with the actinomycete: b) plants whose seeds were treated with a culture of actinomycetes (according to Kublanovskaya (1953).

 

  The crop in these experiments also increased noticeablyin sectors treated with the antagonists. The 8517 variety yielded: 9.4 centners/hain the control sector and 22.6 centners/ha in the experiment. The variety 108-F yielded:17.8 centners/ha in the control sector and in the experiment 22.8 centners/ha.

  Chinese scientists Yin, Chen, Yang et al., (1955) corroboratedKublanovskaya's data. They prepared compost from soil with cotton cake and grew actinomycetesin it which were antagonists of the wilt fungus Verticillium dahliae. Theripened compost was used for the treatment of the seeds. The latter were sown togetherwith the compost. The incidence of the wilt disease decreased by 50-75 %, the cottoncrop increased by 13-45%.

  Mitchell et al, (1948) observed vigorous growth ofantagonists-actinomycetes and bacteria, when those were introduced in the soil togetherwith a composted plant mass. The phytopathogenic fungus causing root rot in the cottonplant perished. The positive role of composted preparations saturated with microbialantagonists was noted by Sanford (1946-1948). He observed in his experiments a dropin the morbidity of potato tubers, pine saplings, etc. Similar data are given bysome other investigators (Weindling, 1946; Garret, 1946; Winter and Rümker,1950). They all noted that the favorable action of composted preparations was notdue to an improvement in plant nutrition but to the more intense development of antagonistsin the soil.

  Schaffnit and Neuman (1953) used peat composts, enrichedwith bacterial antagonists on the snowy mold caused by Fusarium nivale. Thepreparations were made from various types of post. The best results were achievedwith bog peat of mass origin. The percentage of morbidity in wheat treated with compostedpeat was considerably lower than in the control.

  Wood and Tveit (1955), on the basis of data from theliterature and their own observations, came to the conclusion that microbial antagonistsplay an important role in soil improvement. Microbes of local origin are much moreeffective. In order to enhance their growth and activity the authors suggest theintroduction of certain plant residues into the soil as sources of nutrition.

  There are many other studies in the literature whichconfirm the positive effect of microbial antagonists in the struggle against phytopathogenicfungi, bacteria and actinomycetes. All these studies show that microbial antagonistscan be used in agricultural practice for the improvement of soils. For this purposethe soil should be enriched with the appropriate antagonists.

  This can be achieved by various methods. As may beseen from the above data the antagonists mentioned may be introduced into the soilin the form of pure cultures (which is not very effective), or in the form of composts.

  In the enrichment of soils with antagonists, the vegetativecover plays an especially important role. It was noted above that the plants area powerful selective factor. Some of them, under certain conditions favor the growthand accumulation of phytopathogenic microbes in the soil, and others favor the growthof the antagonists of these microbes. By selecting, by means of special experiments,those plants in whose rhizosphere the needed antagonists grow abundantly, and byusing these plants in the crop rotation, one may remove or suppress the growth andthe harmful activity of the pathogenic microbe.

  If the selected plants are inoculated with microbialantagonists before sowing, their accumulation in the soil can thus be markedly enhanced.

  Upon introduction of pure cultures of antagonists,one must take into account their adaptability, their growth in the soil, and theiractivity. If the antagonists lose their activity in the soil or in a substrate whichis not suitable for them which often happens), or if they do not grow or grow butlittle, their effect will be small or will not express itself at all.




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