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Part IV, continued: 

Microflora of decomposing roots

  Investigations have shown that plants not only determinethe microflora of the soil during their growth but also determine them by means oftheir dead residues, especially those of roots. It was established that these residues,depending on the species of plant or, more accurately, on their chemical composition,are decomposed by various forms of microbes. The qualitative composition of the microfloraof the decaying roots of wheat and clover, cotton and lucerne, differ considerably.

  We performed microbiological studies of the root floraand of the flora of the area around roots which were rotting in the soil after theharvesting of crops. Studies have shown that around the roots of wheat, corn, sunflower,soybean, and other plants, the number of microorganisms is considerably higher thanin the soil outside the root zone. If by ordinary calculation (plating on an agarmedium) in the soil outside the root zone one finds four to eight million bacteriain one gram, then around decaying roots there are 20 to 140 million. The number ofbacteria varies according to the degree of the decomposition of the root tissue.

  The group composition of the microflora of decayingroots is given in Table 85, which shows that the increase in the total number ofmicroorganisms paralleled the multiplication of the cellulose bacteria.

Table 85
Qualitative composition of the microflora of corn roots at various stages of decay
(number of cells in millions per gram of soil)

Time of sampling after harvesting of crops, in days

Nonspori- fireous bacteria

Spori- ferious bacteria

Coccoid forms

Myco- bacteria

Actino- mycetes

Fungi

Cellulose bacteria

5

52

6.5

3.5

17.0

9.0

0.3

6.8

12

46

8.5

3.0

12.0

12.0

0.8

5.0

18

120

12.5

2.5

17.5

4.0

0.3

13.0

22

180

15.0

2.8

15.0

5.0

0.6

12.0

27

120

14.0

12.0

27.5

15.0

0.3

8.0

32

100

10.0

20.0

30.0

25.0

1.5

4.0

38

60

5.0

20.0

23.0

22.0

1.6

3.0

  Actinomycetes, mycobacteria, and coccoid forms appearedsomewhat later, after the development of the nonsporiferous bacteria. Coccoid formsare basically mycobacteria, actinomycetes. and, to a certain degree, mycococci, i.e.,organisms belonging to the group of actinomycetes.

  Our subsequent studies were made with root residueswhich were introduced into the soil. We studied the microflora of the decaying rootsof lucerne, clover, wheat, corn, and Euagropyrum, introduced in podsol soil in glassvessels.

  At the same time, the microflora of a root-mass compostwas studied under the conditions of a laboratory experiment. The results are givenin Table 86.

Table 86
Quantitative and qualitative composition of the microflora of decaying roots
(number of cells in thousands per one gram of soil)

Roots

Root- nodule bacteria

Azoto- bacter

Nonspori- ferous bacteria

Mycolytic bacteria

Actino- mycetes

Fungi

Sporiferous bacteria

IN COMPOSTS:

 

 

 

 

 

 

 

Lucerne

1,000,000

0

1,000,000

1,000,000

0.05

1

0.01

Clover

1,500,000

0

1,000,000

10,000

0.1

5

0.001

Wheat

0

0

100,000

0.05

15

35

35

Corn

0.03

0.3

400,000

0.1

250

15

150

Euagropyrum

0.05

0.01

5,000,000

1,000,000

150

20

0.001

 

 

 

 

 

 

 

 

IN SOIL:

 

 

 

 

 

 

 

Lucerne

500,000

2,500

1,200,000

10,000

150

40

0.5

Clover

150,000

1,700

750,000

1,000

300

30

0.8

Wheat

0

0

15,000

0

1,500

360

280

Corn

0

0

13,000

0

4,500

120

340

Euagropyrum

10

0

70,000

100,000

2,800

400

450

  In the table, data are given on a number of microbesduring the initial stage of root decay. During the subsequent stages of decomposition,the quantitative ratios of microorganisms changed; the number of actinomycetes increasedconsiderably, while the number of sporiferous and nonsporiferous bacteria and fungidecreased. In a half-decayed mass of roots, actinomycetes often cover the root particleswith a white coating of aerial mycelium.

  Nonsporiferous bacteria prevail during all stages ofroot decay, but their species composition varies. The number of cellulose bacteriaduring the root decay of various plants differs. Filter paper spread on a soil platecontaining the roots of clover or lucerne decomposes more quickly and more effectivelythan on soil containing the roots of wheat. In the former case, 500-700 eroded spotswere counted on the paper and in the latter case, only 130-270 spots were found.A compost of the roots of Euagropyrum contains fungi which are absent in the decayingroots of clover, and vice verse. In some cases, there is a mass multiplication ofmycolytic bacteria and, in other cases, these bacteria are scarce or not detectedat all (Krasil'nikov and Nikitina, 1945).

  Bodily (1944) introduced residues of clover and wheatstraw into the soil and he observed that in the presence of clover the number ofmicroorganisms in soil was greater than in the presence of wheat straw.

  Other plant residues, in addition to roots, possess,to a certain degree, a determining effect on the composition of the soil microflora.Birch leaves decompose differently and possess a different microflora than leavesof aspen and oak. The composition of the microflora of Euagropyrum straw and wheatstraw also differ. Consequently, all the plant residues of the roots, stems and leavesupon decomposition, enhance the growth of different species of microorganisms inthe soil.

  Al'bitskaya (1954) obtained data on fungi from herstudy of the decomposition of plant residues of forest and steppe vegetation. Accordingto these data, the roots of steppe vegetation are mainly decomposed by fungi of thegenera Penicillium, Cephalosporium , Fusarium, and also, to a limited extent,by members of the genus Mucorales. Roots of oak are decomposed by fungi ofthe genus Trichoderma--T. lignorium, T. koningii, and rarely, by members ofthe genus Penicillium.

  Plant residues of both steppe and forest vegetationare more intensively decomposed after being inoculated by a mixture of the naturalmicroflora where, in these cases, the greatest loss in water-soluble organic substancesis observed, which indicates a most complete decomposition of the residues. Uponthe decomposition of steppe vegetation, there is a greater CO2 evolutionand a decrease in water-soluble substances and in lignin. Upon the decompositionof the roots and leaves of oak, the water-soluble substances are depleted in carbon,and upon the decompo sition of steppe vegetation, their car bon content increases.

  Thus, plants, while alive, differentiate, select, andaccumulate certain compounds. In other words, the vegetative cover, as a whole, isa powerful determining factor in the microbial biocoenoses of soils. In the zoneof the root system, only those organisms which assimilate the root excretions ofthe plant in question more quickly, can develop, supplanting other, less well adapted,species.

  In different plants, the dominating microbial formsdiffer in their systematic position as well as biologically. It may be said thateach species of plant, or group of closely related species, concentrates a more orless specific microflora.

  Unfortunately we are not yet in the position to determinethis specificity in a precise way. Our methods of recognizing and classifying microbes,especially bacteria, are far from being completely developed. We cannot exactly sayin what manner the nonsporiferous bacteria, which dominate in the rhizosphere ofwheat differ from the bacteria in the rhizosphere of clover, oats or potatoes. Bytheir appearance, cell size, motility, colony structure, and nature of growth onmedia, they do not differ in the majority of cases, nor do they differ in their generallyaccepted physiological properties.

  Only a more thorough study of the biochemical activityof microorganisms will enable us to differentiate between them. However, such a methodof study has not yet found wide use in laboratory investigations of rhizosphere microflora.

  The selective action of the vegetative cover may bedirected toward the selection not only of useful, but also of harmful microflora.Under unfavorable conditions, when agrotechnical rules are not observed, with anincorrect choice of crop rotation, the fields are contaminated by phytopathogenicbacteria and fungi and other harmful microbes--weeds. Especially, after the prolongedrepeated cultivation of plants on the same field in monocultures, one observes thiseffect. The accumulation of an undesirable microflora under monocultures is mostoften caused by the insufficient growth of microbial antagonists in the rhizosphere,which are characteristic of the given plant under conditions of normal growth.

  Many outbursts of epiphytotic diseases, as for examplefusarial infections of cereals, cotton, saplings of woody plants, as well as cottonwilt are, in our opinion, caused by the above phenomenon. This was proved by microbiologicalstudies made of cotton fields which were afflicted by the fungi Verticillium dahliaeand Fusarium vasinfectum. These fungi are removed as soon as mycolytic bacteriabegin to grow in the soil. These bacteria, as was shown above, grow under lucerneand certain grass mixtures.

  In agricultural practice from time immemorial, croprotation has been used as one of the methods of increasing crop production. It wasempirically found that with certain crop rotations, not only crop production increased,but disease decreased. It is known that lucerne ameliorates the soil in cotton farmsand inhibits the growth of the organisms causing cotton diseases. On this basis,one can select plants for rational crop rotation.

  Noting the great influence of the vegetative coveron the formation of microbial biocoenoses in soil, one should not forget the importanceof the soil itself as a substrate and the effect of the activity of man on externalconditions. The physicochemical state of soil determines to a great extent the directionof the microbiological processes, and to a similar extent the development of differentmicrobial species. The distribution of Azotobacter in soil not only dependson the plants. on their root excretions and decomposition products, but also on soilacidity and the presence of phosphorus, calcium, molybdenum, and other nutrient elements.

  The cultivation of soil, the liming of soils, the useof fertilizers and other factors favor the growth and accumulation of Azotobacter.In arid regions the growth and accumulation of Azotobacter in soils dependsto a considerable degree on irrigation.

Effect of Soil Microorganisms on Plants

  In the foregoing chapter, the considerable influenceof the microbial population of the soil, and the accumulation of individual speciesand groups of microorganisms in the root zone, was shown. The importance of rootmicroflora for the life of plants has been studied only a little and the informationavailable on the action of different microbes on the growth of plants is meager.We will not dwell here on the activity of root-nodule bacteria, Azotobacter,and mycorhizal fungi, since data on this subject are abundant in scientific literature.In this chapter, data are given only on those organisms which exert a beneficialor harmful effect on plants, due to the products of their metabolism; these are microbialantagonists, activators, inhibitors, etc.

Microbial activators

  It was noted above that certain soil microorganismsare capable of producing various biotic substances--vitamins, auxins, amino acids,and other biocatalysts. Such microorganisms activate the biological processes and,therefore, we named them microbial activators.

  There is much data in the literature on the positiveeffect of pure cultures of bacteria, fungi, and actinomycetes on the growth and developmentof plants. Microbial activators increase the percentage of germinating seeds, enhancethe growth of the young plants, and often change the nature of the biochemical processes.

  Already toward the end of the last century, Geier (1882),and later Zimmermann (1902), described the bacteria living in the tissues of plantswhich had a certain activating effect on their growth. Such bacteria could form specialnodules in the leaves of subtropical and tropical plants.

  According to their systematic position, these bacteriadiffer from each other. In members of the genus Ardisia Thunb., the nodulesin the leaf tissues are formed by nonsporiferous bacteria of the genera Bacteriumand Pseudomonus. In Pavetta L., Chomelia L,, PsychotriaL., and certain other genera, mycobacteria were isolated from the nodules, in DioscoreaL., and others, bacteria of the Rhizobium type were isolated (Krasil'nikov,1940 a, b).

  Miehe (1911, 1918) studied in detail the bacteria whichare members of the genera Ardisia Thunb. and Pavetta L. According tohis data, they formed special substances which cause the stimulation of the tissues(Reizwirkung). They do not fix nitrogen.

  Jongh (1938) found that Ardisia does not growor grows poorly without bacterial symbionts, and does not bloom nor bear fruit. Whenbacteria were introduced into the tissue of the plant, its growth and developmentimproved drastically, the growth of branches was enhanced, leaves acquired normalform, and flowers and fruits appeared.

  The role of the symbionts of the root-nodule bacteriagroup is widely known. Forming nodules on the roots of leguminous plants and on certainnonleguminous ones, under certain conditions they considerably improve the growthof plants and increase crop yields.

  The biological role of root-nodule bacteria for plantsis widely known. However, the mechanism of the action of these organisms is stillobscure. It is assumed that root-nodule bacteria fix molecular nitrogen and supplyit to the host plant. There is no clear-cut experimental data to support this assumption.

  There are reasons to believe that root-nodule bacteria,as well as the bacteria from the nodules on the leaves of the above-mentioned plants,act favorably through their metabolites. According to our data, leguminous plants,in symbiosis with the nodule hacteria, fix molecular nitrogen for themselves fromthe air. The bacteria, due to their metabolic products, act as biocatalysts, activatingthe nitrogen-fixing ability (Krasil'nikov and Korenyako, 1946a).

  The positive action of mycorhizal fungi has alreadybeen mentioned. These fungi are widespread in nature. One view has been expressedthat all plants have mycorhizae, but differ as to the nature of the co-habitation.In some plants the mycorhiza is endotrophic and in others ectotraphic. In the former,the fungal hyphae grow almost exclusively in the root tissues and only a few extendinto the soil, outside the root, The endotrophic mycorhiza, in its turn, includestwo types of mycorhizae the phycomycetal and vesicular type, wore often encounteredin grassy and woody plants, and the orchid mycorhiza found in the plants of the orchidfamily These two types of mycorhiza differ in the nature of the structure and developmentof their mycelial hyphae. In phycomycetal mycorhizas, the hyphae are not septateand often form characteristic swellings in the root tissues--vesiculae. The myceliumin the orchid mycorhiza is septate; the hyphae form characteristic entanglementsonly within the root cello. As a rule, they do not have vesicles.

  In the endotrophic mycorhiza of both types, the hyphaedevelop only in the cortex of roots in the intercellular space, or penetrate intothe cells. The mycelia of the fungus do not penetrate into the central part of theroot.

  Ectotrophic mycorhiza is characterized by the growthof mycelial hyphae on the surface of the root tips, which surrounds them with a thickand quite dense cover. From this cover the hyphae extend into the soil. There areno root hairs on this part of the root. A small part of the hyphae penetrates insidethe root, but not very deeply, their growth being usually limited to the intercellularspace of the epidermis, where the hyphae interweave, forming he dense Hartig net.The hyphae seldom penetrate into the upper two to three layers of the cortical cells.The hyphae do not penetrate the cortical cells, and, in the few cases where thisdoes happen, they soon die inside the cells. Entotrophic mycorhizae are most oftenencounter ed among woody coniferous and leaf-bearing plants.

  Between the endotrophic and ectotrophic mycorhizae,there are intermediate formations--the ecto-endotrophic mycorhizas.

  Some authors (Lobanov, 1953) are of the opinion thatan absolutely ectotrophic mycorhiza does not exist at all. They maintain that inwoody plants, especially during the early stages of their development, there is alwaysan endotrophic mycorhiza. Only later the external cover on the root tip develops.

  The systematic distribution of the mycorhiza fungivaries. Fungi-forming vesicular mycorhiza probably belong to Phycomycetes of theEndogenaceae family, The endotrophic mycorhizas in orchids are formed by the FungiImperfecti of the genus Rhizoctonia, while in certain orchids, the mycorhizais ferried by higher fungi, Basidiomycetes with well-developed fruiting bodies--Armillariamellea, Xerotus javanicus, and Marasmius coniatus.

  Ectotrophic and endotrophic mycorhizae of woody plantsare mainly formed by mushrooms, most often by the imperfect fungi of the genus Phoma.There are mycorhiza-forming fungi, among the Ascomycetes and other groups of fungi.For instance, in beech, 12 different species of fungi have been described as participantsin the mycorhizae in pine 17 species were described; and, in spruce, up to nine species,etc (Kursanov, 1940; Yachevskii, 1933; Kelly, 1952; Magru, 1949; Lilly and Barnett,1953, Goimann, 1954, Lobanov, 1953 and others).

  These data clearly show that there is no strict specificityamong mycorhizal fungi, as in root-nodule bacteria. The first to observe the positiveeffect of mycorhizal fungi on the growth of plants was Kamenskil (1880) and afterhim, Voronin (1886), Vysotskii (1902), and others. They all looked upon mycorhizalfungi as symbionts, which exert a great influence on the growth of plants. Baraney(1940) presents extensive data confirming this point of view. One of his tables isgiven below (Table 87).

Table 87
Effect of mycorhizas on the growth of pine seedlings

Indexes of growth

With mycorhizas

Without mycrohizas

Length of shoots of seedlings, cm

35.5

17.5

Increase in length after 2 years, cm

18.0

3.0

Length of sprouts of 2nd order, cm

10.0

0.3

Weight of shoots part, gm

17.0

3.1

Weight of roots, gm

11.0

4.5

Number of leaves

42

12

Total area of leaves, cm2

591.0

96.0

  The essence of the action of fungi consists in supplyingthe plants with nitrogenous and carbonaceous elements of nutrition in some casesand, in others, in the supply of auxiliary nutrients or biotic substances, and morecorrectly with both. There is a great deal of data in the literature on the significanceof mycorhizal fungi in the nutrition of plants, which was already mentioned In theprevious chapters of this work. Recently, by the use of direct experiments with labeledatoms, it was shown that mycorhizal fungi take up find transmit various nutrientelements.

  Kramer and Wilbur (1949) and Mellin and Nilson (1950,1952) have shown that fungi transmit P32 and N15 from the externalsolution into the tissues of the roots and stems of pine (Pinus taeda L.,P. resinosa, P. silvestris L.). Morrison (1954) found that in the presenceof the mycorhizas, there in enhanced transfer of P32, not only to theroots and atoms of Pinus Rediata, but also to the leaves. Herley and MacCready(1950, 1952) have shown that mycorhizal fungi take up labeled phosphorus, accumulatingup to 90% in their mycelia.

  The data on studies made with labeled atoms does notdisclose the nature of the compounds by way of which the labeled phosphorus and nitrogenare transmitted to the mycorhizal fungi, one must assume that these elements whenentering the cell of the fungus, take part in the general process of building itssubstance in the form of one of the organic compounds of metabolic products, Thelabeled elements are released from the cell as metabolites which enter into the mediumand, from there, into the roots and green parts of the host plant. Such it processwas demonstrated in Shavlovskii's experiment with rhizosphere bacteria (see above).

  The free-living soil bacteria also have a considerableeffect on plants. Clark and Roller (1931) studied the action of pure cultures ofthe following bacteria on the growth of duckweed: Bact. coli, Clostridium sporogenes,Clastrid welchii, Ps. fluorescens liquefaciens, Bact. aerogenes, Staph. aureus, Bac.subtilis, Bact. prodigiosum etc. Some of these bacteria stimulated the growthof buckwheat, while others had no visible effect on it.

  Kozlowski (1935) observed the action of pure bacteriacultures on the growth of barley and apples, The tested cultures of the sporferousbacteria, Bac. cereus, Bac. mycoides, and Bac. subtills and of thenonsporiferous bacteria, Bact. denitrificans, Bact. putidium, and Ps. pyocyaneaand also cultures of fungi. The sporiferous bacteria had no effect on the growthof plants. Of the nonsporiferous bacteria, Ps. pyocyanea and Bact. putiduminhibited their growth, while Bact. denitrificans, stimulated the growth ofbarley, but not that of apples.

  We tested 130 different bacteria, mycobacteria, andactinomycetes isolated from various soils. Among them were the following; 32 strainsof Azotobacter, 33 strains of root-nodule bacteria, 40 of Pseudomonas,10 of Bacterium, four of mycobacteria, six of actinomycetes, three of Bac.mycoides, and two of Bac. subtilis. Cultures of these organisms and productsof their metabolism were added to the medium in which wheat seedlings were grownin one series of experiments, and to that of isolated roots of peas and wheat inanother series of experiments.

  Isolated roots are a convenient object for the studyof the requirements of biotic substances, since they are incapable of the independentsynthesis of the whole gamut of these substances.

  We grew isolated roots of peas, wheat, rye and otherplants on Bonner's synthetic medium of the following composition:

Distilled water 1,000 ml
Ca (N03) 2 . 4H2 O, 0.23 g
MgSO4 . 7H2O, 0.36 g
KNO3,,0.81 g
KCl, 0.65g
KH2PO4,0.12 g
Iron, Traces
Saccharose, 20 g

  Extracts and filtrates of bacterial cultures were addedin various amounts, as auxiliary substances. The bacterial cultures were grown inliquid media and filtered with bacterial filters. The results are given in Table88.

Table 88
Influence of metabolic products of soil microorganisms on the growth of isolated roots
(increase in cm on the 30--40th day of growth)

Microorganisms

Increase in length of roots of peas

Increase in length of roots of wheat

Control (no metabolites of microbes)

0.3

0.1

Az. chroococcum

 

 

strain 54

15.0

17.0

strain 37

5.0

0.8

strain A

27.0

15.0

Rh. terifolii

 

 

leguminosarum

31.0

30.0

phaseoli

50.1

45.0

lupini

7.0

15.0

meliloti

0.0

5.0

sojae

3.0

10.0

Ps. aurantiaca

65.0

55.0

Ps. flourescens

 

 

strain 4

40.0

35.0

strain 15

10.0

0.5

strain 30

25.0

12.0

strain 69

38.0

12.0

Ps. denitrificans

6.0

38.0

Ps. mycolytica

10.0

31.0

Ps. nonflourescens

 

 

strain 3

4.0

30.0

strain 10

10.0

31.0

strain 12

23.0

11.0

strain 15

0.1

0.2

Bact. denitrificans

25.0

12.0

Bact. album

37.0

37.0

Bact. mycolyticum

6.0

25.0

Bact. vulgaris

0.0

0.0

Bac. mycoides

0.0

5.0

Bac. mesentericus

 

 

strain 3

0.0

3.0

strain 11

0.0

0

strain 27

5.0

0

strain 29

3.0

0

Bac. subtilis

 

 

strain 7

0.0

0

strain 17

2.0

0

strain 21

3.0

0

A. violaceus

0.0

0

A. aurantiacus

3.0

0

A. globisporus

 

 

strain 160

65.0

25.0

strain 187

37.0

30.0

strain 375

10.0

45.0

A. grisus

 

 

strain 17

0.0

0.0

strain 57

0.0

0.0

strain 1067

0.0

25.0

A. albus

37.0

56.0

A. alboflavus

71.0

45.0

  As can be seen from the table, a large increase inthe length of the roots was observed in the presence of filtrates of Azotobacter,root-nodule bacteria, and bacteria of the genera Pseudomonas and Bacterium(Figure 82) Metabolic products of certain actinomycetes were quite active. Sporiferousbacteria often showed a negative action, inhibiting the growth of roots.

 

Figure 82. Effect of products of bacterial metabolism on the growth of isolated roots of peas:

1--metabolites of Ps. fluorescens ; 2--metabolites of Ps. aurantiaca; 3-control (no metabolites added).

 

  In the same microbial group and even in the same species,different strains showed different effects on the roots. For example, among culturesof Ps. fluorescens, strain No 4 strongly activates the growth of wheat roots,while strain No 15 only slightly activates or does not activate these roots at all;strainNo 15 of Ps. nonfluorescens is inactive, while strains Nos 5 and 10are active; the root-nodule bacteria of lucerne did not promote the growth of theseroots and even suppressed them, while the root-nodule bacteria of peas and especiallythose of beans greatly enhanced root growth. The same was observed in all other groupsof organisms.

  The roots of different plants reacted differently tothe action of filtrates of the same culture. The roots of wheat reacted more intensivelythan the roots of peas to the metabolites of Ps. nonfluorescens, strain 10,while with the filtrate of strain 12, the picture was reversed.

  Filtrates of microbial cultures show a positive effectonly when used in small amounts. When the amounts are large, their effect on thegrowth of isolated roots is negative. The roots do not grow or grow very poorly,deviating from the normal, they thicken, swell, do not branch, become brown too soon,and die.

  The metabolites of many organisms in small concentrationsstrongly suppress the growth of roots. Such organisms which produce toxic substancesare found among various groups of microorganisms but they are especially numerousamong the sporiferous bacteria. This group of bacteria is in general the most toxicin relation to plants and many microbes (see below).

  Similar data were obtained in experiments with plantseedlings. The filtrates of certain microorganisms noticeably activated the germinationof seeds, while the filtrates of others had an inhibitory or no effect.

  As in the experiments with isolated roots, filtratesof Azotobacter, nonsporiferous bacteria of the genera Pseudomonas, Rhizobiumand Bacterium, and many species of actinomycetes most actively enhanced thegrowth of seedlings, In the genus Azotobacter, the strains of Az. vinelandiiand Az. agile var. jakutiae were active. The strains of Az. chroococcumdiffered considerably in activity. Some possessed strongly and accentuated activatingproperties with respect to the growth of wheat, others had only weak activating propertiesand still others had no effect whatsoever on the growth of plants. Under the influenceof certain strains, the suppression of wheat growth was observed.

  We studied the activating effect of bacteria on differentplants under field conditions for a period of three years (1945a). The seeds weretreated with a culture of bacterial activators and were sown on kolkhoz fields invarious regions. Altogether more than 100 experiments were performed, not includingthose with Azotobacter. The summary of the results is given in Table 89,

Table 89
Effect of bacterial activators on plant crop

Crop

Bacteria

number of experiments

positive experiments

increase in crops, %

Wheat  

 

 

 

  Ps. flourescens

 

 

 

  strain No. 14

16

12

12-27

  strain No. 30

9

7

15-25

  strain No. 25

12

6

10-14

  Bact. sp strain No. 106

10

8

15-29

  Oligonitrophiles

10

7

12-18

  Az. chroococcum strain No. 103

20

12

10-23

Oat  

 

 

 

  Ps. flourescens

 

 

 

  strain No. 14

10

7

14-28

  strain No. 30

--

--

--

  strain No. 25

5

5

10-16

  Bact. sp strain No. 106

12

8

12-30

  Oligonitrophiles

2

2

14-22

  Az. chroococcum strain No. 103

25

15

13-19

Clover  

 

 

 

  Ps. flourescens

 

 

 

  strain No. 14

6

5

18-23

  strain No. 30

4

2

12-25

  strain No. 25

4

3

10-30

  Bact. sp strain No. 106

6

4

18-26

  Oligonitrophiles

--

--

--

  Az. chroococcum strain No. 103

12

8

14-27

  The data in Table 89 show that bacterial activatorsexert a similar effect on crops as do azotogen*, nitrogen, and other bacterial compounds.In our experiments, Azotobacter was used in the form of peat azotogen. *[*Azotogen--Russiancommercial name for azotobacter field-inoculating preparation.] In Table 89are given only those cases where a positive effect was obtained when Azotobacterwas absent from the soil; it had perished during the first few days after its introductioninto the soil. Therefore, the effect was caused, not by Azotobacter, but byother microbes.

  Akhromeiko and Shestakova (1954) successfully testedbacteria, which had been isolated from the rhizosphere, on the growth of oak andash tree seedlings. With oak, there was a 24-34 per cent increase in the incrementof dry matter, and with ash a 40 per cent increase. Samtsevich and others (1952)used an Azotobacter culture for the inoculation of oak seedlings in a steppezone. According to their observations, this microbe increases the percentage of acorngermination and enhances the growth of oak seedlings. Similar results were obtainedby Runov and Enikeeva (1955), Mishustin (1950b) Smali (1951) and others.

  Shtern (1940 a, b) tested radiation strains of Azotobacteron oat seedlings, grown in vegetation containers. Certain radiation strains weremore active than the initial culture.

  Afrikyan (1954a) studied a large collection (more than200 strains) of sporiferous bacteria isolated from Armenian soils. The wheat seedswhich were inoculated with these cultures were allowed to germinate either in Kochdishes on cotton or in sand in containers. The experiments showed that there arevery few activators among the sporiferous bacteria of the Bac. subtilis andBac. mesentericus group. More often one finds bacterial inhibitors in thisgroup which suppress seed germination and the growth of plants. These data are inagreement with our observations (see below).

  Popova (1954) employed cultures of bacteria isolatedfrom the rhizospere of grape vines for the enhancement of the germination of grapeseeds and grape stalks. Certain species of Ps. sinuosa increased the percentageof germinating seeds to 80% while in the control plants, only 10-12% of the seedsgerminated by the 45th day. These bacteria also enhanced the growth of seedlingsand roots. In the control plants, the buds swelled on the 16th day and, in thosetreated with bacteria, on the fourth day. The highest activity was shown by Azotobacterchroococcum and the nonsporiferous bacterium Bact. album, strains 2 and3.

  Pantosh (1955) found that nonsporiferous bacteria ofthe Pseudomonas and Bacterium groups have an activating effect on the growth of theplant from the rhizosphere of which they were isolated. In experiments performedin vegetation containers, on quartz sand, the inoculation of the seeds with bacteriabefore sowing increased a crop of wheat by 20-65%above that of the controls, as follows:

Inoculated with

Weight of dry plants, g

Total nitrogen content, mg

In the control (uninoculated)

9.9

376

Bacterium sp.

11.1

430.7

Ps. radiobact.

16.5

466.5

Flavobacterium solare

11.2

408.9

Bact. parvulum

14.3

430.3

  Petrosyan (1956) studied the effect of bacterial activatorson leguminous plants, on the formation of their nodules and on their accumulationof nitrogen. The experiments were performed in containers and in plots under fieldconditions. In the former case, the following results were obtained:

 

Weight of plants, g

Number of nodules

Total nitrogen, %

In experiments with vetch

 

 

 

Control (no bacteria added)

34 (100%)

110

4.86

Inoculated with root-nodule bacteria

45 (132%)

121

5.11

Inoculated with rood-nodule bacteria and activator

65 (190%)

161

5.29

Inoculated with a pure culture of activator

56 (163%)

129

5.88

 

 

 

 

In experiments with lucerne

 

 

 

Control (no bacteria added)

34 (100%)

87

4.39

Inoculated with root-nodule bacteria

38 (109%)

129

4.96

Inoculated with rood-nodule bacteria and activator

53 (153%)

177

5.24

Inoculated with a pure culture of activator

48 (139%)

155

5.07

  Similar results were obtained in field experiments.

  The increase in crops due to bacterial activators wasas follows: after vetch, 172.6%--control crop of 11.6 kg; after lucerne, 141.3%--controlcrop of 10.2 kg; after Onobrychis, 150%--control crop of 8.4 kg from one plot.

  In our experiments, we obtained similar results. Invegetation containers certain bacterial activators (Ps. aurantiaca No 1, PseudomonasNo 145, Bacterium sp. No 160 produced an increase in leguminous plants asfollows: clover, beans, lucerne and lupine, by 30-80% above that of the control plants.In field experiments the crop increase of these plants after inoculating them withthe activators, was 24-30% above the control levels. The number of nodules increasedconsiderably when activators were employed.

  On the roots of one plant, the following number ofnodules were found: in control plants without inoculation of bacteria, 8; on plantsinoculated with root-nodule bacteria of lucerne, 9-12; and after inoculation of bacterialactivators (Ps. aurantiaca), 28. On the roots of beans, the number of noduleswas 6. 8 and 16; on the roots of lupine, 0.2, 0.5 and 1.2 (Krasil'nikov and Korenyako,1945c).

Inoculation of seeds

Dry mass of clover obtained, g

Uninoculated

26

Active initial culture

28

Avirulent strain I

35

Avirulent strain II

21

Avirulent strain A

40

Avirulent strain D

42

Avirulent strain B

26

Avirulent strain A1

36

Avirulent strain A3

26

Avirulent strain E

18

  Bacterial activators stimulate the activity of root-nodulebacteria, enhancing the formation of nodules and, by means of the latter, the growthof the plants. Activators also act positively on leguminous plants without nodulebacteria directly stimulating their growth by their metabolic products.

  In one series of experiments, we employed avirulentstrains of the root-nodule bacteria of clover and vetch, obtained experimentally.The seeds were inoculated with cultures of these bacteria and sown in sterile sandin glass containers. Crops were obtained after two months.

 

Figure 83. Organotropic action of bacteria on lucerne:

Exp. A: a--stimulation of root growth with the simultaneous suppression of the growth of the aerial parts; b--control plants; exp. B: a--stimulation of the growth of aerial parts; b--control plants.

 

 

Figure 84. Effect of metabolic products of bacteria on the growth of Phycomyces blakeslseanus:

1--control growth of the fungus in the absence of metabolites; 2--mass formation of zygotes (dark spots) in the presence of metabolites of bacterial stimulators (Bacter. sp. No 2); 3--abundant growth of aerial mycelium with conidia in the presence of metabolites of bacterial activators (Azotobacter chroococcum).

 

  Similar results were obtained in experiments with vetch.Using avirulent experimental strains of root-nodule bacteria, Nos 1, A, D, and A1,the crop was considerably greater (123-161%) than when the inoculation was performedwith the initial virulent culture (107-115%). In experiments with avirulent bacteria,no nodules were found on the roots of the plants while when the initial culture wasused for Inoculation, 9-58 nodules were found on each plant.

  Certain chemically pure substances obtained from culturesof actinomycetes and other fungi, as for instance gibberellins and gibberellin-likesubstances, activate the growth of plants (Figure 85).

 

Figure 85. Effect of antibiotics on the growth of corn:

1--seeds treated with antibiotic solution before sowing, 2--control, seeds treated with water.

 

  Dorosinskii and Lazarev (1949), Dorosinskii (1953),Lazarev and Dorosinskii (1953) grew oats in sterile, well-washed, loamy soil in thepresence of bacteria and in their absence.

  The plant crop in the absence of bacteria but withfull mineral fertilization was, on the average, 2.6 g; in vessels with bacteria,7.1 g; in vessels with bacteria, but without a mineral fertilizer, 6.5 g.

  Fomin (1951) grow certain melon cultures, fruit, andwood varieties of plants, treating them with preparations of Azotobacter,Psaudomonas, and "silicate" bacteria. After such treatment, thecrops of all these plants increased.

  Certain microorganisms exert an organotropic actionon plants: they activate the growth of individual organs or tissues. For instance,Ps. tumefaciens stimulates the multiplication of the cells of the root tissueor stem tissue in tomatoes, carrots, and other plants, as a result of which swellingsare formed, There are microbes which, by their metabolic products only, activateto a considerable extent, the growth of roots or serial parts.

  In our investigations, we have observed bacterial cultureswhich, under the conditions of vegetation experiments (in sand) only stimulated thegrowth of roots, not affecting the aerial parts at all. In other variations of theexperiment some bacteria enhanced the growth of the aerial parts without influencingthe root system (Figures 83 and 85). Observations were also made of bacterial cultureswhich activate the process of fertilization in fungi (Figure 84) and yeasts, In Table90, data are given on the activity of the substances which stimulate this process.

Table 90
Activity of substances stimulating the sexual process of fungi and yeasts
(number of units in 1 g of dry substance)

Substrate

Phycomyces blakesleanus

Zygosacchar. sp.

Compost inoculated with bacteria

150

80

Soil humus

60

60

Az. chroococcum strain A

180

120

Ps. flourescens strain 30

150

30

Extract from an aspen raceme

260

80

  Molliard (1903) observed the stimulation of the formationof apothecia in the fungus Ascobolus under the influence of the bacteria within.Sartory (1916) obtained perithecia in aspergilli only in those comes where the fungigrow together with the sporiferous bacillus Bac. mesentericus.

  Stimulation of the copulation of the fungus Phycomycesblakealesanus when grown together with bacteria or their metabolic products (factorZ), extracted from agar-agar, was observed by Robbins and Schmidt (1939, 1945). This"factor", according to their data, is present in the tissues of many plantsand is also formed by microbes.

  Nickerson and Thimann (1941, 1943) found that a certainsubstance among the metabolic products of the fungus Aspergillus niger enhancesthe copulation of the yeast Zygosaccharomyces. The active principle of thisstimulant is soluble in water and 90% ethyl alcohol and consists of two substances:an acid closely related to glutamic acid, and riboflavin, Burnett (1956) caused anincreased copulation and the formation of zygotes in mucor fungi by the additionof ß-carotene to the medium.

  In our collection, there are actinomycete-antagonists,the metabolic products of which have a stimulating effect on the fruiting processesin higher plants. One such culture enhanced flowering and fruition in the cottonplant. Under conditions of the experiment in growth containers, plants treated withthe native liquid of the given actinomycete had 10-12 bolls, while the control plantshad 5-6 bolls on each bush. Correspondingly, the cotton crop approximately doubledin quantity. We treated plants with nonpurified substances. It should be assumedthat chemically purified preparations will have an even stronger activating effecton plants.

  In recent years great interest has arisen over gibberellinsand, especially, gibberellic acid, as a stimulant to the growth and development ofplants. These substances are obtained from the fungus Gibberella fujikoroi(a conidial stage of Fusarium moniliforme). This fungus was first isolatedin Japan by Kurozava, in 1926, from the tissues of diseased rice. The rice diseasecaused by this fungus is quite widespread in Japan, and expresses itself in an extremeelongation of the stems, in the yellowing of leaves, and in the death of the plant.Yabuta isolated the active substance from a culture of the fungus and he found thatit stimulated the growth of many plants. Later, this substance was isolated in thecrystalline form and studied in greater detail. In England and in America, threesubstances, gibberellin A1, gibberellin A2, and gibberellicacid (gibberellin A3) were obtained from the culture fluid. The last substanceis the most active and is of the greatest interest. It was, therefore, more thoroughlystudied and more fully elucidated, Chemically defined, it is a dihydroxylacetoneacid, a tetracyclic compound, with the general formula C19H22O6.

  The activating effect of gibberellic acid expressesitself in extremely small concentrations. Only 0.01 microgram/ml is enough to stimulatethe growth of peas; at higher concentrations the stimulation of growth increases.The greatest effect is observed at a concentration of 10 micrograms/ml. Accordingto the data obtained by Brian (1957), the increment in the growth of peas exceedsby 10 times that of the control plants. Its height was 42 cm after treatment withgibberellic acid, while in the control, nontreated plants, it was 7 cm. This stimulatingeffect was also observed by Brian in experiments with wheat, but to a lesser degreethan in the experiments with peas. Gibberellic acid does not show an activating effecton the growth of roots, neither in peas nor in wheat.

  Of special interest is the effect of gibberellic acidon the growth of biennial plants: cabbage, rape, carrots, sugar beets, etc. It isknown that these plants give off flower shoots and bear fruit during their secondyear of life; during their fire, year of growth only a rosette of leaves and rootsis formed. The flowering and formation of seeds may also be achieved during the firstyear of growth, but only after yarovization [a Russian term for vernalization; translator].

  Experiments have shown that these plants form flowershoots and flowers and produce seed during their first year of growth after beingtreated with gibberellic acid, without previous yarovization, This acid has the sameeffect as the one obtained by yarovization. The enhancement of flowering and fruitionis observed in longday plants. Lang (1956) obtained flowers and fruit on henbane(Hyoscyamus niger) during the first year of its growth. After the introductionof 300 micrograms/ml in the tissue, the plant soon formed flowering, or main shoots,on which flowers developed.

  During the last two or three years, a vast amount ofmaterial has accumulated showing the strong activating effect of gibberellic acidon the growth and flowering of various plants. The strongest effect is seen withthe long-day plants.

  It should be noted that the stimulating effect of gibberellicacid was so far only obtained under experimental conditions in a hothouse. Underfield conditions in soil, there have either been no results, or a weak stimulationof only certain plants has been observed. A small increase of crops, within the rangeof 11-25 per cent has been observed in meadow grasses.

  In plants which react to gibberellic acids, one oftenobserves that there is a decrease in the amount of chlorophyll, that the leaves havea yellowish tint, that their total nitrogen content decreases, and that in the tobaccoleaves the percentage of nicotine decreases and , in rice, a decrease in the amountof sugars. It is assumed that this is caused by a lack of nutrition. With appropriatefertilization this has not been observed.

  The mechanism of the action of gibberellic acid isnot clear; however, all the investigators note that it differs from that of auxins.The latter affects the processes of growth in a different way. These substances alsodiffer in their chemical composition.

  The data given in this chapter show that there arespecies of microorganisms which form very active substances, which stimulate thegrowth and certain processes and functions in plants. Gibberellic acid is the firstmetabolic product of microorganisms obtained in a chemically pure form. It shouldbe assumed that in the near future many other substances will be obtained which possessthe capacity to activate the growth and development of plants. Similarly, as takesplace among microbial antagonists. The stimulating substances of the microbial activatorsbelong to various classes of compounds. Here, a great research study lies ahead inthe isolation of these substances and in the study of their nature.

  One must note that among the antibiotic substancesand activating compounds, there is often much in common in the way they act on organisms.Many antibiotics show a stimulating effect on the growth of plants and animals; theyenhance the increment of live weight of the latter, and sometimes enhance the processof fruition in both higher and lower plants. On the other hand, activating substancesquite often possess clearly expressed antimicrobial properties.

Distribution of microbial activators in the soil

  Very little is known on the distribution and growthof microbial activators in soil. At the same time, in daily laboratory practice,one very often encounters these bacteria, We have in mind the auxoautotrophs, whichhave been mentioned before. These organisms synthesize all the substances necessaryfor growth and development, and, therefore, grow well on simple synthetic media.For the purpose of their study, we used the following medium:

Twice distilled water 1,000 mm
KNO3 1.0 g
KH2PO4 1.0 g
MgSO4 . 7 H20 0.2 g
CaCl2 0.1 g
NaCl 0.1 g
FeCl3 traces
Glucose 20.0 g

  There were no vitamins or auxins in the medium.

  The total number of auxoautotrophs growing on thismedium was quite large. We counted from several tens of thousands to many hundredsof millions of bacteria in one gram of soil.

  The number of auxoautotrophic bacteria changes, dependingon the properties of the soil and climatic conditions. As a rule, their total numberis greater in fertile soils than in nonfertile ones. When the auxoautotrophs fromthe chernozem soils of Moldavia, Crimea, and Kuban are plated on an agar medium,30 to 150 million appear in one gram. In the turfy podsol soils of the nonchernozembelt of the Moscow and Leningrad Oblast's and of Latvia, the number of these organismsvaries within the range of 100,000 to 4.5 million per gram. In the soils of the KolaPeninsula (primary soils, loose calcareous soils, etc) there are 5,000 to 30,000cells in one gram,

  In cultivated soils their number is greater than innoncultivated soils, and in gardens it is greater than in fields (Table 91).


Table 91
Quantitative ratio of auxoautotrophic and auxoheterotrophic types in soils
(in thousands per one gram of soil)

Soil and region

On a vitamin-free medium

On a meat-peptone agar MPA

Podsol. Arctic Circle. Virgin soil

5

3.5

Podsol. Arctic Circle. Cultivated soil

300

360

Podsol. Moscow Oblast'. Virgin soil

200

180

Podsol. Moscow Oblast'. Cultivated soil

2,500

2,000

Podsol. Moscow Oblast'. Garden soil

45,000

60,000

Krasnozem. Caucasus. Cultivated soil

3,500

1,500

Podsol. Latvia. Virgin soil

150

100

Podsol. Latvia. Cultivated soil

1,800

1,500

Chernozem. Moldavia. Virgin soil

1,200

800

Chernozem. Moldavia. Cultivated soil

30,000

50,000

Chernozem. Crimea. Virgin soil

20,000

35,000

Chernozem. Crimea. Cultivated soil

160,000

200,000

Chernozem. Kuban'. Cultivated soil

220,000

280,000

Chernozem. Kuban'. Cultivated soil

450,000

600,000

  Comparing the data on the number of bacteria growingon a synthetic vitamin-free medium and on an ordinary protein medium, one can observethat the number of auxoautotrophs and auxoheterotrophs is almost the same. In humussoil the ratio between these two categories is approximately 1:1. In certain cases,there are even more auxoautotrophs (see above).

  According to Schmidt and Starkey (1951), about 30 percent of soil bacteria synthesize biotic substances and excrete them into the soil.

  Lochhead and Chase (1943) found from 10-14 per centof auxoautotrophs among the microflora of soil. These authors divide soil microbesinto seven groups, according to their ability to grow on vitamin-free media withthe addition of a few auxiliary substances. To the first group belong the microbesgrowing on media completely devoid of vitamins. In the second group are includedorganisms growing on a vitamin-free medium with the addition of a few amino acids(cysteine, alanine, proline, asparagine, arginine, leucine, glycine, lysine, etc).To the third group the authors relate those microbes which require for growth certainvitamins: pantothenic and nicotinic acids, thiamine, riboflavin or others. The fourthgroup includes organisms growing on a medium to which both amino acids and vitaminswere added. Those microorganisms growing on a basal vitamin-free medium with an admixtureof yeast extract comprise the fifth group.

  A basal vitamin-free medium with an admixture of soilextract reveals the bacteria of the sixth group. This medium, with a small admixtureof yeast and soil extracts, is suitable for the bacteria of the seventh group, themost numerous one. Microbes of the first group comprise approximately 10-14 per centof all soil bacteria; the second group, 10 per cent; the third, 12-14 per cent; thefourth 16-17 per cent; the fifth, 18-20 per cent; the sixth, 3-7 per cent; and finally,the seventh, 40-50 per cent of all soil bacteria. This subdivision of bacteria isquite arbitrary.

  A great number of vitamin-producing bacteria are foundin the rhizosphere of plants. According to our calculations, they comprise 40-80per cent of all soil bacteria and their number varies depending on the species ofthe plant, the stage of its growth, and upon external conditions. In analyzing therhizosphere of wheat and clover, growing in the fields of the Moscow district Dolgoprudnoe),we obtained the data given in Table 92. In the root zone of these two plants andin all other plants studied by us, the number of auxoautotrophs was no smaller andoften larger than the number of auxoheterotrophs.

Table 92
Number of auxoautotrophic bacteria in the rhizosphere of plants
(in thousands per gram of soil)

Plant

Soil

Autxoauto- trophs in early phase of growth

Auxohetero- trophs in early phase of growth

Auxoauto- trophs in the fruit-bearing phase

Auxohetero- trophs iin the fruit-bearing phase

Wheat Rhizosphere

800,000

100,000

300,000

500,000

Wheat Control

5,000

3,500

3,000

3,000

Clover Rhizosphere

1,500,000

1,800,000

1,000,000

1,600,000

Clover Control

40,000

30,000

20,000

20,000

  West and Lochhead (1940a, b), and Wallace and Lochhead(1949), in their detailed studies also found that auxoautotrophs prevailed in therhizosphere of flax and other plants. The same was noted by Katznelson and Richardson(1943), Stolp (1952) and others (Table 93).

Table 93
Quantitative ratios of auxoautotrophs in the rhizosphere and outside it, by percentage
(according to Wallace and Lochhead, 1949)

Plant and soil

Early growth stage

Flowering stage

Wheat / Rhizosphere

42.3

48.5

Wheat / Outside the rhizosphere

14.4

9.7

Oats / Rhizosphere

44.1

47.4

Oats / Outside the rhizosphere

14.8

9.7

Clover / Rhizosphere

55.5

42.8

Clover / Outside the rhizosphere

9.3

2.4

Lucerne / Rhizosphere

40.5

37.2

Lucerne / Outside the rhizosphere

9.3

2.4

Flax / Rhizosphere

35.8

32.9

Flax / Ouitside the rhizosphere

9.7

8.6

Timothy grass / Rhizosphere

25.3

15.4

Timothy grass / Outside the rhizosphere

9.3

2.4

  As is well known, the ability of microorganisms toproduce biotic substances is, in most cases, not connected with their taxonomic divisioninto groups. Auxoautotrophs and heteroauxotrophs are found among various microbialgroups: among sporiferous and nonsporiferous bacteria, micrococci, mycobacteria,actinomycetes, fungi, etc. The largest percentage is encountered among the bacteriaof the genus Pseudomonas. According to our calculations, the number of thesebacteria reaches 0-60 per cent of all the bacteria growing on an agar medium devoidof vitamins. A smaller, but quite a high percentage (30-40 per cent) is encounteredamong the genus Bacterium and mycobacteria. Considerably rarer are the auxoautotrophsamong sporeforming bacteria of the genus Bacillus (Table 94).

Table 94
Number of auxoautotrophs of different groups of microorganisms,
living in the rhizosphere of plants and outside the root zone
(by percentage)

Bacteria

Outside the rhizosphere

In the rhizosphere, wheat

In the rhizosphere, clover

Pseudomonas

30.0

40.0

46.0

Bacterium

40.0

34.0

23.4

Mycobacterium

18.0

25.0

30.0

Bacillus

6.0

0.3

0.1

  Oligonitrophiles are active producers of biotic substances.These organisms are widely encountered in soils and their characteristic featureis that they grow abundantly in a vitamin-free and nitrogen-free medium. Evidently,they all belong to the auxoautotrophs.

  Among the actinomycetes one rarely finds cultures whichwill not grow on Chapek' s synthetic medium. They grow well on a vitamin-free medium.

  Yeasts of the genera Torula, Mycotorula, andsome others are widespread in the soil. These organisms are powerful producers ofvitamins and various other biotic substances. Therefore, they grow well on mineral,vitamin-free media, forming large slimy or semislimy colonies. Some of them, as,for instance, Torulopsis pulcherrima are widespread in the soil and grow abundantlyon the nitrogen-free medium of Ashby, forming large colonies similar to Azotobactercolonies. They are of special interest as activators of plant growth and of the lifeprocesses of microorganisms.

 


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