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Nodule bacteria and pea symbiosis. Nodule bacteria are symbiotic nitrogen-fixing organisms.

The first soil bacteria that humanity noticed were nodule bacteria. About 1300 out of 13 thousand plants form a nodule, and in agriculture 200 are used. Of these, all have the function of fixing atmospheric nitrogen. In the soil on the nodule, microorganisms settle and multiply - symbionts that replace fertilizers.

What is nodule bacteria

More than 2 thousand years ago, farmers noticed that poor, exhausted soil resources yield crops after cultivation on them legumes. The next attempts to reveal the secret were in 1838: J.-B. Boussengo decided that the leaves of legumes fix nitrogen, but experiments with an unfavorable aquatic environment didn't confirm it. In 1901, Azotobacter chroococcum (6 species from the genus Azotobacter) was discovered. The first drug based on "earthen" bacteria Nitragin was created in 1897.

All nodule bacteria are microaerophiles. They have a rod-shaped/oval shape. Rhizobium (Rhizobiales) belong to those capable of converting the gaseous form of nitrogen into a plant-assimilable - soluble. Data:

By the extent to which microorganisms affect the crop, they are divided into active (effectively enrich the soil), inactive and inactive (inefficient).
When there is no moisture, they do not reproduce, therefore, in a dry climate, specially infected plants are introduced deeper into the soil.
The optimal temperature for reproduction of all representatives of nitrogen-fixing organisms is 20-30°C, but growth continues at 0-35°C. The best environment (pH) is neutral, about 6.5-7.1, but acidic causes the death of colonies.
Thanks to the experiments of the Moscow Agricultural Academy, it turned out that even in the absence of "donors", the bacterial material does not leave the soil for up to 50 years.
Microorganisms are able to survive even conditions after atomic explosion, withstand gamma radiation and ultraviolet, solar radiation, but can not inhabit under high temperature.
Microorganisms are of maximum importance for root development.

The role of nodule bacteria in nature

In addition to the fixation of atmospheric nitrogen, the role of nodule bacteria in nature is very large. In the process of reproduction, they "engage" in the synthesis of vitamins, natural antibiotics, and contribute to the development of the root first, and then the tops. The benefit lies in the fact that soil bacteria of the nitrogen-fixing type due to symbiosis with plants:

are part of the cycle of matter - nitrogen;
synthesize phytohormones, stimulating plant growth;
can be used as a way of self-purification of soils contaminated with heavy metals under mineralizing factors (natural / enterprises);
decompose some chlorine-containing compounds.

Legumes and nodule bacteria

through tissue damage
penetration through the root hairs;
penetration through young root tips;
thanks to companion bacteria.

Symbiotic bacteria of the genus Rhizobium, having penetrated the root, move into its tissues, easily overcoming the intercellular space in groups or single cells (like in lupine). More often, during reproduction, the cell forms infectious threads (strands, colonies). Their number varies by plant type. Often there are common threads of infection, forming a single nodule.

Nitrogen fixation by bacteria

The value that nitrogen fixation by bacteria represents is enormous: it not only restores the soil, but also allows you to get richer crops than on humus or chemical fertilizers. There is an interaction between the substance and the nitrogen fixer:

in Azotobacter (“autonomous”, not requiring the presence of a plant) - by enzymes, due to oxygen in the cell;
in Rhizobium (nodule bacteria) - only in the presence of magnesium, sulfur, iron.

Paleontological evidence suggests that some plants belonging to the Eucaesalpinioideae group were the most ancient legumes that had nodules.

At modern species leguminous plants nodules are found on the roots of many members of the Papilionaceae family.

Phylogenetically more primitive representatives of such families as Caesalpiniaceae, Mimosaceae, in most cases do not form nodules.

Of the 13,000 species (550 genera) of leguminous plants, the presence of nodules has so far been identified only in approximately 1300 species (243 genera). This primarily includes plant species used in agriculture (more than 200).

Having formed nodules, legumes acquire the ability to absorb atmospheric nitrogen. However, they are able to feed on bound forms of nitrogen - ammonium salts and nitric acid. Only one plant, Hedysarum coronarium, assimilates only molecular nitrogen. Therefore, without nodules in nature, this plant does not occur.

Nodule bacteria supply the leguminous plant with nitrogen, which is fixed from the air. Plants, in turn, supply bacteria with carbohydrate metabolism products and mineral salts that they need for growth and development.

In 1866, the famous botanist and soil scientist M.S. Voronin saw the smallest “bodies” in the nodules on the roots of leguminous plants. Voronin put forward bold assumptions for that time: he linked the formation of nodules with the activity of bacteria, and the increased division of root tissue cells with the reaction of the plant to bacteria that penetrated the root.

20 years later, the Dutch scientist Beijerin isolated bacteria from the nodules of peas, vetch, chiny, beans, seradella and lollipop and studied their properties, checking the ability to infect plants and cause the formation of nodules. He named these microorganisms Bacillus radicicola. Since bacteria that form spores belong to the genus Bacillus, and nodule bacteria lack this ability, A. Prazhmovsky renamed them Bacterium radicicola. B. Frank proposed a more successful generic name for nodule bacteria - Rhizobium (from the Greek rhizo - root, bio - life; life on the roots). This name has taken root and is still used in the literature.

To designate a species of nodule bacteria, it is customary to add a term corresponding to the generic name Rhizobium Latin name of the plant species from whose nodules they are isolated and on which they can form nodules. For example, Rhizobium trifolii - clover nodule bacteria, Rhizobium lupini - lupine nodule bacteria, etc. In cases where nodule bacteria are able to form nodules on the roots different types leguminous plants, that is, to cause the so-called cross-infection, the species name is, as it were, a collective one - it is precisely this “cross-infecting” ability that is reflected in it. For example, Rhizobium leguminosarum - nodule bacteria of peas (Pisum), lentils (Lens), ranks (Lathyrus).

Morphology and physiology of nodule bacteria. Nodule bacteria are characterized by an amazing variety of forms - polymorphism. Many researchers drew attention to this when studying nodule bacteria in pure culture in laboratory conditions and soil. Nodule bacteria can be rod-shaped and oval. Among these bacteria there are also filterable forms, L-forms, coccoid immobile and mobile organisms.

Young nodule bacteria in pure culture on nutrient media usually have a rod-shaped shape (Fig. 143, 2, 3), the size of the rods is approximately 0.5-0.9 X 1.2-3.0 microns, mobile, multiply by division. In rod-shaped cells of nodule bacteria of clover, division by ligation is observed. With age, rod-shaped cells can move to budding. According to Gram, the cells stain negatively, their ultrafine structure is typical of gram-negative bacteria (Fig. 143, 4).

With aging, nodule bacteria lose their mobility and pass into the state of the so-called girdled rods. They got this name due to the alternation of dense and loose sections of protoplasm in the cells. The striation of the cells is well detected when viewed under a light microscope after treatment of the cells with aniline dyes. Dense sections of protoplasm (girdle) are stained worse than the spaces between them. In a luminescent microscope, the bands are light green, the spaces between them do not glow and look dark (Fig. 143, 1). Belts can be located in the middle of the cage or at the ends. The zonation of the cells is also visible on electron diffraction patterns, if the preparation is not treated with contrast agents before viewing (Fig. 143, 3). Probably, with age, the bacterial cell is filled with fatty inclusions that do not perceive color and, as a result, cause the cell to be striated. The stage of "belted rods" precedes the stage of formation of bacteroids - irregularly shaped cells: thickened, branched, spherical, pear-shaped and flask-shaped (Fig. 144). The term "bacteroids" was introduced into the literature by J. Brunhorst in 1885, applying it to formations of unusual shape, much larger than rod-shaped bacterial cells, found in nodule tissues.

Bacteroids contain large quantity volutin granules and are characterized by a higher content of glycogen and fat than rod-shaped cells. Bacteroides grown in artificial nutrient media and formed in nodule tissues are physiologically of the same type. It is believed that bacteroids are forms of bacteria with an incomplete division process. With incomplete cell division of nodule bacteria, dichotomously branching forms of bacteroids arise. The number of bacteroids increases with the aging of the culture; their appearance is facilitated by the depletion of the nutrient medium, the accumulation of metabolic products, and the introduction of alkaloids into the medium.

In old (two-month-old) cultures of nodule bacteria, using an electron microscope, it is possible to identify clearly defined spherical formations in many cells (Fig. 145) - arthrospores. Their number in cells varies from 1 to 5.

On nutrient media, nodule bacteria of various types of leguminous plants grow with different speed. Fast-growing bacteria include rhizobia of peas, clover, alfalfa, fodder beans, vetch, lentils, chiny, sweet clover, fenugreek, beans, chickpeas, and bird-foot; to slow-growing - nodule bacteria of lupine, soybean, peanut, seradella, mung bean, cowpea, sainfoin, gorse. Fully formed colonies of fast-growing cultures can be obtained on the 3rd - 4th day of incubation, colonies of slow-growing cultures - on the 7th - 8th.

Fast-growing nodule bacteria are characterized by a peritrichous arrangement of flagella, while slow-growing bacteria are monotrichial (Table 42, 1-5).

In addition to flagella, filamentous and bead-like outgrowths are formed in cells of nodule bacteria when grown on liquid media (Tables 42, 43). Their length reaches 8-10 microns. They are usually located on the surface of the cell peritrichially, they contain from 4 to 10 or more per cell.

Colonies of fast-growing nodule bacteria have the color of baked milk, often translucent, slimy, with smooth edges, moderately convex, and eventually grow on the surface of the agar medium. Colonies of slow-growing bacteria are more convex, small, dry, dense and, as a rule, do not grow on the surface of the medium. The mucus produced by nodule bacteria is a complex compound of the polysaccharide type, which includes hexoses, pentoses and uronic acids.

Nodule bacteria are microaerophiles (they develop with small amounts of oxygen in the environment), however, they prefer aerobic conditions.

Nodule bacteria use carbohydrates and organic acids as a carbon source in nutrient media, and various mineral and organic nitrogen-containing compounds as a nitrogen source. When cultivated on media with a high content of nitrogen-containing substances, nodule bacteria may lose their ability to penetrate the plant and form nodules. Therefore, nodule bacteria are usually grown on plant extracts (bean, pea broth) or soil extracts. The phosphorus necessary for development can be obtained by nodule bacteria from mineral and organic phosphorus-containing compounds; mineral compounds can serve as a source of calcium, potassium and other mineral elements.

To suppress extraneous saprophytic microflora when isolating nodule bacteria from nodules or directly from the soil, nutrient media with the addition of crystal violet, tannin or antibiotics are recommended.

For the development of most cultures of nodule bacteria, an optimum temperature is required in the range of 24-26 °. At 0° and 37°C growth stops. Usually cultures of nodule bacteria are stored in laboratory conditions at low temperatures (2-4 °C).

Many types of nodule bacteria are capable of synthesizing B vitamins, as well as growth substances such as heteroauxin (-indoleacetic acid).

All nodule bacteria are approximately equally resistant to an alkaline reaction of the medium (pH = 8.0), but unequally sensitive to an acidic one.

Specificity, virulence, competitiveness and activity of nodule bacteria.

concept specificity nodule bacteria - collective. It characterizes the ability of bacteria to form nodules in plants. If we talk about nodule bacteria in general, then for them the formation of nodules only in a group of leguminous plants is already specific in itself - they have selectivity for leguminous plants.

However, if we consider individual cultures of nodule bacteria, it turns out that among them there are those that are able to infect only a certain, sometimes larger, sometimes smaller, group of leguminous plants, and in this sense, the specificity of nodule bacteria is a selective ability in relation to the host plant. The specificity of nodule bacteria can be narrow (clover nodule bacteria infect only a group of clovers - species specificity, and lupine nodule bacteria can even be characterized by varietal specificity - infect only alkaloid or alkaloid-free varieties of lupine). With a wide specificity, pea nodule bacteria can infect pea, chin, and bean plants, and pea and bean nodule bacteria can infect pea plants, i.e., they are all characterized by the ability to “cross-infect”. The specificity of nodule bacteria underlies their classification.

The specificity of nodule bacteria arose as a result of their long-term adaptation to one plant or to a group of them and the genetic transmission of this property. In this regard, there is also a different adaptability of nodule bacteria to plants within the cross-infection group. Thus, alfalfa nodule bacteria can form nodules in sweet clover. Nevertheless, they are more adapted to alfalfa, and sweet clover bacteria are more adapted to sweet clover.

In the process of infection of the root system of leguminous plants with nodule bacteria great importance It has virulence microorganisms. If specificity determines the spectrum of action of bacteria, then the virulence of nodule bacteria characterizes the activity of their action within this spectrum. Virulence refers to the ability of nodule bacteria to penetrate the root tissue, multiply there, and cause the formation of nodules.

An important role is played not only by the very ability to penetrate into the roots of the plant, but also by the speed of this penetration.

To determine the virulence of a strain of nodule bacteria, it is necessary to establish its ability to cause the formation of nodules. The criterion for the virulence of any strain can be the minimum number of bacteria that provides a more vigorous infection of the roots compared to other strains, culminating in the formation of nodules.

In soil, in the presence of other strains, the more virulent strain will not always infect the plant first. In this case, one should take into account competitive ability, which often masks the property of virulence in natural conditions.

It is necessary that virulent strains also have competitiveness, i.e., they can successfully compete not only with representatives of the local saprophytic microflora, but also with other strains of nodule bacteria. An indicator of the competitiveness of a strain is the number of nodules formed by it as a percentage of the total number of nodules on plant roots.

An important property of nodule bacteria is their activity(efficiency), i.e., the ability to assimilate molecular nitrogen in symbiosis with leguminous plants and satisfy the needs of the host plant in it. Depending on the extent to which nodule bacteria contribute to an increase in the yield of legumes (Fig. 146), they are usually divided into active (effective), inactive (ineffective) and inactive (ineffective).

A strain of bacteria inactive for one host plant in symbiosis with another species of leguminous plant can be quite effective. Therefore, when characterizing a strain in terms of its effectiveness, it should always be indicated in relation to which host plant species its effect is manifested.

The activity of nodule bacteria is not their permanent property. Often in laboratory practice, there is a loss of activity in cultures of nodule bacteria. In this case, either the activity of the entire culture is lost, or individual cells with low activity appear. A decrease in the activity of nodule bacteria occurs in the presence of certain antibiotics, amino acids. One of the reasons for the loss of activity of nodule bacteria may be the influence of the phage. By passaging, i.e., repeatedly passing bacteria through the host plant (adaptation to a particular plant species), it is possible to obtain effective strains from ineffective ones.

Exposure to y-rays makes it possible to obtain strains with enhanced efficiency. There are known cases of the emergence of highly active radiomutants of alfalfa nodule bacteria from an inactive strain. The use of ionizing radiation, which has a direct effect on the change in the genetic characteristics of the cell, in all likelihood, can be a promising technique in the selection of highly active strains of nodule bacteria.

Infection of a leguminous plant with nodule bacteria.

To ensure the normal process of infection of the root system with nodule bacteria, it is necessary to have a fairly a large number viable bacterial cells in the root zone. The opinions of researchers regarding the number of cells required to ensure the process of inoculation are different. Thus, according to the American scientist O. Allen (1966), 500-1000 cells are required for the inoculation of small-seeded plants, and at least 70,000 cells per 1 seed are required for the inoculation of large-seeded plants. According to the Australian researcher J. Vincent (1966), at the moment of inoculation, each seed should contain at least several hundred viable and active nodule bacteria cells. There is evidence that single cells can also penetrate into the root tissue.

During the development of the root system of a leguminous plant, the multiplication of nodule bacteria on the root surface is stimulated by root secretions. The destruction products of root caps and hairs also play an important role in providing nodule bacteria with a suitable substrate.

In the rhizosphere of a leguminous plant, the development of nodule bacteria is sharply stimulated; for cereal plants, this phenomenon is not observed.

On the surface of the root there is a layer of mucous substance (matrix), which is formed regardless of the presence of bacteria in the rhizosphere. This layer is clearly visible when examined in a light-optical microscope (Fig. 147). Nodule bacteria after inoculation usually rush to this layer and accumulate in it (Fig. 148) due to the stimulating effect of the root, which manifests itself even at a distance of up to 30 mm.

During this period, preceding the introduction of nodule bacteria into the root tissue, the bacteria in the rhizosphere are extremely mobile. IN early work, in which a light microscope was used for research, nodule bacteria located in the rhizosphere zone were given the name of schwermers (gonidia or zoospores) - “swarming”. Using the method of Faereus (1957), it is possible to observe the formation of extremely fast moving colonies of schwermers in the region of the root tip and root hairs. Schwermer colonies exist very a short time- less than a day.

About the penetration mechanism nodule bacteria in the plant root there are a number of hypotheses. The most interesting of them are the following. The authors of one of the hypotheses state that nodule bacteria penetrate into the root through damage to the epidermal and cortical tissue (especially in the places where lateral roots branch off). This hypothesis was put forward on the basis of research by Bril (1888), who caused the formation of nodules in leguminous plants by piercing the roots with a needle previously immersed in a suspension of nodule bacteria. As a special case, such an implementation path is quite real. For example, in peanut nodules are predominantly located in the axils of root branches, which suggests the penetration of nodule bacteria into the root through gaps during the germination of lateral roots.

An interesting and not unfounded hypothesis is the penetration of nodule bacteria into the root tissue through the root hairs. The passage of nodule bacteria through root hairs is recognized by most researchers.

The suggestion of P. Dart and F. Mercer (1965) is very convincing that nodule bacteria penetrate into the root in the form of small (0.1-0.4 µm) coccoid cells through intervals (0.3-0.4 µm) of cellulose fibrillar network of the primary sheath of root hairs. Electron microscopic photographs (Fig. 149) of the root surface, obtained by the replica method, and the fact of shrinkage of nodule bacteria cells in the rhizosphere of leguminous plants confirm this position.

It is possible that nodule bacteria can penetrate into the root through the epidermal cells of young root tips. According to Prazhmovsky (1889), bacteria can penetrate the root only through the young cell wall (of root hairs or epidermal cells) and are completely unable to overcome the chemically altered or corky layer of the cortex. This may explain that nodules usually develop on young sections of the main root and emerging lateral roots.

Recently, the auxin hypothesis has received great popularity. The authors of this hypothesis believe that nodule bacteria penetrate the root due to stimulation of the synthesis of β-indoleacetic acid (heteroauxin) from tryptophan, which is always present in plant root secretions. Fig. 150).

The source of β-indolylacetic acid at the time of infection of the plant, obviously, is not only plants that secrete through root system tryptophan, which many types of bacteria, including nodule bacteria, can convert into β-indoleacetic acid. Nodule bacteria themselves, and possibly other types of soil microorganisms living in the root zone, can also participate in the synthesis of heteroauxin.

However, the auxin hypothesis cannot be accepted unconditionally. The action of heteroauxin is nonspecific and causes curvature of root hairs in various plant species, not just legumes. At the same time, nodule bacteria cause curvature of root hairs only in leguminous plants, while exhibiting rather significant selectivity. If the considered effect were determined only by β-indolylacetic acid, then there would be no such specificity. In addition, the nature of changes in root hairs under the influence of nodule bacteria is somewhat different than under the influence of heteroauxin.

It should also be noted that in some cases, uncurved root hairs are exposed to infection. Observations show that in alfalfa and peas, 60-70% of root hairs are twisted and twisted, and in clover - about 50%. In some species of clover, this reaction is noted in no more than 1/4 of the infected hairs. In the reaction of curvature, obviously, the state of the root hair is of great importance. Growing root hairs are most sensitive to the action of substances produced by bacteria.

Nodule bacteria are known to cause softening of the walls of root hairs. However, they do not form either cellulase or pectinolytic enzymes. In this regard, it was suggested that nodule bacteria penetrate the root due to the secretion of mucus of a polysaccharide nature, which causes the synthesis of the polygalacturonase enzyme by plants. This enzyme, destroying pectin substances, affects the shell of root hairs, making it more plastic and permeable. In small amounts, polygalacturonase is always present in root hairs and, apparently, causing partial dissolution of the corresponding components of the membrane, allows the cell to stretch.

Some researchers believe that nodule bacteria penetrate into the root thanks to satellite bacteria that produce pectinolytic enzymes. This hypothesis was put forward on the basis of the following facts. When microscopy of root hairs, many researchers noted the presence of a light spot, near which nodule bacteria accumulate. This spot, possibly, is a sign of the beginning of maceration (destruction) of the tissue by protopectinase, by analogy with the same sign observed in plants with many bacterial diseases. In addition, it was found that avirulent cultures of nodule bacteria in the presence of bacteria producing pectinolytic enzymes become able to penetrate into the root.

Another hypothesis should be noted, according to which nodule bacteria enter the root during the formation of a finger-like protrusion on the surface of the root hair. The electron diffraction pattern of a root hair section confirming this hypothesis (Fig. 150, 3) shows a root hair bent in the form of an umbrella handle, in the bend of which there is an accumulation of nodule bacteria. Nodule bacteria are as if drawn in (swallowed) by the root hair (similar to pinocytosis).

The invagination hypothesis essentially cannot be separated from the auxin or enzymatic hypothesis, since intussusception occurs as a result of exposure to either an auxin or enzymatic factor.

In some cases it is very difficult to detect the boundaries between phases. The most intensive introduction of nodule bacteria into root hairs occurs at the early stages of plant development. The second phase ends during the mass formation of nodules. Often, the penetration of nodule bacteria into root hairs continues even after the nodules have formed on the roots. This so-called excess or additional infection occurs because the infection of the hairs does not stop. long time. In more late dates infection nodules are usually placed lower down the root.

The type of development, structure, and density of root hairs do not affect the rate of introduction of nodule bacteria. The sites of nodule formation are not always associated with the locations of infected hairs.

Having penetrated into the root (through the root hair, epidermal cell, places of root damage), nodule bacteria then move into the tissues of the plant root. Most easily, bacteria pass through the intercellular spaces.

Either a single cell or a group of bacterial cells can penetrate into the root tissue. If a separate cell has been introduced, it can continue to move through the tissue as a single cell. The way of root infection by single cells is characteristic of lupine plants.

However, in most cases, the invading cell, actively multiplying, forms the so-called infection threads (or infection cords) and, already in the form of such threads, moves into the plant tissues.

The term "infection thread" arose on the basis of the study of the infection process in a light microscope. Beginning with Beijerinck's work, the infection thread came to be seen as a slimy, hyphae-like mass containing proliferating bacteria.

Essentially, an infection thread is a colony of multiplied bacteria. Its beginning is the place where a single cell or group of cells has penetrated. It is possible that a colony of bacteria (and, consequently, a future infection thread) begins to form on the surface of the root before the introduction of bacteria into the root.

The number of infected root hairs varies considerably between individual plants. Usually infectious threads appear in deformed, twisted root hairs. However, there are indications that similar threads are sometimes found in straight hairs. More often, one branching thread is observed in the root hairs, less often two. In some cases, there are several threads in one root hair, or in several there are common threads of infection, giving rise to one nodule (Fig. 151).

The percentage of infected root hairs in the total number of deformed hairs is inexplicably low. It usually ranges from 0.6 to 3.2, occasionally reaching 8.0. The proportion of successful infections is even lower, since among the infectious threads there are many (up to 80%) so-called abortive threads that have ceased to develop. The rate of advancement of normally developing infectious threads in a plant is 5-8 microns per hour. At this speed, the path through the root hair 100-200 microns long can pass through the infection thread within one day.

Morphological and anatomical characteristics of nodules in their ontogenesis.

According to the method of formation, the nodules of leguminous plants are divided into two types:

1st type - nodules arise during the division of pericycle cells (root layer), usually located against the protoxylem (first in time for the formation of vessels) - endogenous type of nodule formation;

Type 2 - nodules originate from the root cortex as a result of the introduction of the pathogen into the parenchymal cells of the cortex and endoderm (the inner layer of the primary cortex) - an exogenous type of nodule formation.

In nature, the latter type predominates. The tissues of the central cylinder of the root take part only in the formation of the vascular system of nodules of both endogenous and exogenous types.

Despite different views on the nature of the origin of nodules of exo- and endotypes, the process of their development is basically the same. However, neither one nor the other type of nodule formation should in any case be identified with the process of formation of lateral roots, despite the fact that there are some similarities in their inception. Thus, the formation of nodules and lateral roots occurs simultaneously and, moreover, in the same root zone.

At the same time, a number of features in the development of lateral roots and nodules emphasize the profound differences in the type of their formation. Lateral roots arise in the pericycle. From the very first moments of development, they are connected with the central cylinder of the main root, from which the central cylinders of the lateral roots branch off, and they always arise against the ray of the primary wood. The formation of a nodule, in contrast to the lateral root, is possible anywhere. At the very beginning of the formation of nodule tissue, there is no vascular connection with the central cylinder of the root; it arises later. Vessels usually form along the periphery of the nodule. They are connected with the root vessels through the tracheid zone and have their own endoderm (Fig. 152).

The difference in the nature of nodulation and lateral roots is especially clearly observed in seradella, since the cortical tissue of the main root of this plant - the site of the first nodules - consists of a relatively small layer of cells and nodules become visible very quickly after infection of the root with bacteria. They first form flattened protrusions on the root, which makes it possible to distinguish them from the conical protrusions of the lateral roots. Nodules differ from lateral roots in a number of anatomical features: the absence of a central cylinder, root caps and epidermis, and the presence of a significant layer of bark covering the nodule.

The formation of nodules (Fig. 153, 1, 2) of leguminous plants occurs during the period when the root still has a primary structure. It begins with the division of cortical cells located at a distance of 2-3 layers from the ends of the infectious threads. The layers of the cortex, penetrated by infectious threads, remain unchanged. At the same time, in seradella, division of cortical cells occurs directly under the infected root hair, while in peas, cell division is noted only in the penultimate layer of the cortex.

Division with the formation of a radial tissue structure continues to the inner core cells. It occurs without a certain direction, randomly, and as a result of this, a meristem (a system of educational tissues) of the nodule arises, consisting of small granular cells.

Divided cells of the cortex change: the nuclei are rounded and increase in size, especially the nucleoli. After mitosis, the nuclei disperse and, without taking their original form, begin to divide again.

A secondary meristem appears. Soon, in the endoderm and pericycle, signs of incipient division appear, which in the former outer cells occurs mainly by tangential septa. This division finally extends to the common meristematic complex, the small cells of which are elongated, the vacuoles disappear, the nucleus fills most cells. The so-called primary nodule is formed, in the plasma of cells of which nodule bacteria are absent, since at this stage they are still inside the infection threads. While the primary nodule is being formed, infection threads branch out many times and can pass either between cells - intercellularly (Fig. 154), or through cells - intracellularly - and introduce bacteria (Fig. 155).

Intercellular infectious threads, due to the active reproduction of nodule bacteria in them, often acquire a bizarre shape - they form in the form of pockets (diverticula) or torches (see Fig. 154).

The process of movement of infection threads from cell to cell is not entirely clear. Apparently, infectious threads, as Canadian microbiologist D. Jordan (1963) believes, wander in the form of naked mucous strands in the intercellular spaces of plant tissue until, due to some still inexplicable reasons, they begin to invaginate into the cytoplasm of adjacent cells.

Distribution of nodule bacteria in nature

Being symbiotic organisms, nodule bacteria spread in soils, accompanying certain types of leguminous plants. After the destruction of the nodules, the cells of nodule bacteria enter the soil and begin to exist at the expense of various organic substances, like other soil microorganisms. The almost ubiquitous distribution of nodule bacteria is evidence of their high degree of adaptability to various soil and climatic conditions, the ability to lead a symbiotic and saprophytic way of life.

Schematizing the currently available data on the distribution of nodule bacteria in nature, we can make the following generalizations.

In virgin and cultivated soils, nodule bacteria of those species of leguminous plants that are found in the wild flora or have been cultivated for a long time in a given area are usually present in large quantities. The number of nodule bacteria is always the highest in the rhizosphere of leguminous plants, somewhat less in the rhizosphere of other species, and few in the soil away from the roots.

Both effective and ineffective nodule bacteria are found in soils. There is a lot of evidence that the long-term saprophytic existence of nodule bacteria, especially in soils with unfavorable properties (acidic, saline), leads to a decrease and even loss of bacterial activity.

Rice. 1 - Nodules on alder roots (according to J. Becking)

Cross-infection of different species of leguminous plants often leads in nature and agricultural practice to the appearance of nodules on the roots that do not actively fix molecular nitrogen. This, as a rule, depends on the absence of the corresponding types of nodule bacteria in the soil.

This phenomenon is especially often observed when using new species of leguminous plants, which are either infected with ineffective species of Cross-group bacteria or develop without nodules.

Rice. 2 - Nodules on the roots of tribulus (according to O. Allen)

Nodule bacteria are used for industrial production nitragin, used to treat the seeds of legumes. They were first discovered by M. S. Voronin in 1866. Later, M. V. Beyerink (1888) isolated them in pure culture and studied in detail by microbiologists and physiologists. Bacteria enter the roots of leguminous plants through the root hair and penetrate into the inner integument of the root, into the parenchyma, causing increased cell division and proliferation. On the roots, ugly growths are formed, called nodules, or nodules. At first, the bacteria absorb the nutrients of the plant and somewhat inhibit its growth. Then, as the nodule tissue grows, a symbiosis is established between bacteria and higher plants. Bacteria receive carbonaceous food (sugar) and minerals from the plant, and in return provide it with nitrogenous compounds.

Nodule bacteria settle in the soil, multiply, and penetrate the root cells through holes in the root hairs of leguminous plants. In the cells there is an increased reproduction of nodule bacteria and in parallel there is an intensive division of root cells infected with nodule bacteria.

Nodule bacteria supply the leguminous plant with nitrogen. The plant utilizes this bound nitrogen and in turn supplies the nodule bacteria with the carbon-containing organic matter they need. Nodule bacteria can use various sugars and alcohols as a carbon source.

Nodule bacteria are microaerophiles (they develop with small amounts of oxygen in the environment), however, they prefer aerobic conditions. Nodule bacteria that have emerged from the infection thread continue to multiply in the host tissue. The bulk of bacteria multiply in the cytoplasm of the cell, and not in the infection thread. They develop most intensively when the soil reaction is close to neutral. Therefore, when sowing legumes on acidic soils along with seed inoculation, soil liming is necessary. Inoculation without liming has very little effect on yield and protein content.

Nodule bacteria are capable of favorable conditions in one season to accumulate up to 200 - 300 kg / ha of nitrogen.

Young nodule bacteria in pure culture on nutrient media usually have a rod-shaped shape (Fig. 2, 3), the size of the rods is approximately 0 5 - 0 9 X 1 2 - 3 0 μm, mobile, multiply by division

In addition to nodule bacteria, other microorganisms that can assimilate free nitrogen from the air also live in the soil; they do not live on the roots of plants, but near them. All other nutrients needed by these microbes, they absorb on their own, and not at the expense of plant juices, as is inherent in nodule plants. Azotobacter is the most important microorganism living in the soil, capable of assimilating atmospheric nitrogen. These bacteria can live under favorable conditions of humidity, good air flow, suitable temperature and acidity of the soil. Azotobacter requirements for thermal regime and soil moisture are about the same as the requirements of cultivated plants, but it is more sensitive to soil acidity than most plants.

Nodules are formed only in representatives of the legume family (Fabaceae). IN various plants nodules differ only in shape and size. They are formed after penetration into the root system of nodule bacteria.

Numerous studies have shown that nodule bacteria differ from each other, and therefore the genus Rhizobium should be considered as a group of related microorganisms. At a young age, these bacteria are motile, rod-shaped, 1.2 to 3 μm in length, the placement of flagella in some species is peritrichous, in others - subpolar. Nodule bacteria are Gram-negative, non-spore-bearing aerobic organisms.

Aging, nodule bacteria lose their flagella, cease to be mobile and take on the form of girdled rods, since with age the bacterial cell is filled with fatty inclusions and does not stain. With aging, thickened, branched, spherical and other forms of formation often appear in the nodules of the Rhizobium culture, much larger than ordinary cells. These polymorphic formations are called bacteroids.

Nodule bacteria can assimilate various carbohydrates, organic acids, and polyhydric alcohols. Amino acids are available to them as a source of nitrogen. For most Rhizobium crops optimal value The pH of the medium is 6.5-7.5, and the optimum temperature is 24-26 ° C.

It has been established that nodule bacteria can infect only a certain group of leguminous plants. The selective ability of these bacteria with respect to plants is called specificity. This property has become the main feature for the development of the taxonomy of nodule bacteria.

In some cases, not only species, but also varietal specificity of nodule bacteria is observed. In addition to specificity, these bacteria are characterized by virulence - the ability to penetrate the root tissue, multiply there and cause the formation of bubbles. Under certain conditions, these bacteria can reduce or even lose their activity.

An essential property of nodule bacteria is also their activity, that is, the ability to assimilate molecular nitrogen in symbiosis with plants. Strains of active and inactive nodule bacteria occur in the soil. Infection of leguminous plants with an active race of bacteria leads to the formation of a large number of bubbles on the main root and causes a vigorous process of atmospheric nitrogen fixation. Inactive races of these bacteria cause bubbles to form, but nitrogen is not fixed.

Nodules, which are formed by active races of bacteria, are pink in color. The pigment that gives them this color is similar in chemical composition to blood hemoglobin and is called leghemoglobin (phytoglobin). It is believed that this pigment contributes to the process of nitrogen assimilation, maintaining the redox potential at a certain level. Nodules that form inactive races of bacteria are greenish in color.

Paleontological evidence suggests that some plants belonging to the Eucaesalpinioideae group were the most ancient legumes that had nodules.

In modern species of leguminous plants, nodules have been found on the roots of many representatives of the family Papilijonaceae.

Phylogenetically more primitive representatives of such families as Caesalpiniaceae Mimosaceae do not form nodules in most cases.

In 1866, the famous botanist and soil scientist M.S. Voronin saw the smallest "calves" in the nodules on the roots of leguminous plants. Voronin put forward bold assumptions for that time: he linked the formation of nodules with the activity of bacteria, and the increased division of root tissue cells with the reaction of the plant to bacteria that penetrated the root.

20 years later, the Dutch scientist Beijerinck isolated bacteria from the nodules of peas, vetch, chiny, beans, seradella and lollipop and studied their properties, checking the ability to infect plants and cause the formation of nodules. He named these microorganisms Bacillus radicicola. Since bacteria that form spores belong to the genus Bacillus, and nodule bacteria lack this ability, A. Prazhmovsky renamed them Bacterium radicicola. B. Frank proposed a more successful generic name for nodule bacteria - Rhizobium (from the Greek rhizo - root, bio - life; life on the roots). This name has taken root and is still used in the literature.

To designate a species of nodule bacteria, it is customary to add a term to the generic name Rhizobium corresponding to the Latin name of the plant species from whose nodules they are isolated and on which they can form nodules. For example, Rhizobium trifolii - clover nodule bacteria, Rhizobium lupini - lupine nodule bacteria, etc. In cases where nodule bacteria are able to form nodules on the roots of different types of leguminous plants, i.e., cause the so-called cross-infection, the species name is, as it were, collective - it reflects precisely this “cross-infecting” ability. For example, Rhizobium leguminosarum - nodule bacteria of peas (Pisum), lentils (Lens), ranks (Lathyrus).

Morphology and physiology of nodule bacteria - nodule bacteria are characterized by an amazing variety of forms - polymorphism. Many researchers drew attention to this when studying nodule bacteria in pure culture in laboratory conditions and soil. Nodule bacteria can be rod-shaped and oval. Among these bacteria there are also filterable forms, L-forms, coccoid immobile and mobile organisms.

Young nodule bacteria in pure culture on nutrient media usually have a rod-shaped shape, the size of the rods is approximately 0.5-0.9 X 1.2-3.0 microns, mobile, multiply by division. In rod-shaped cells of nodule bacteria of clover, division by ligation is observed. With age, rod-shaped cells can move to budding. According to Gram, the cells stain negatively, their ultrafine structure is typical of Gram-negative bacteria.

With aging, nodule bacteria lose their mobility and pass into the state of the so-called girdled rods. They got this name due to the alternation of dense and loose sections of protoplasm in the cells. The striation of the cells is well detected when viewed under a light microscope after treatment of the cells with aniline dyes. Dense sections of protoplasm (girdle) are stained worse than the spaces between them. In a fluorescent microscope, the bands are light green, the spaces between them do not glow and look dark. Bands can be located in the middle of the cell or at the ends. The girdling of cells is also visible on electron diffraction patterns, if the preparation is not treated with contrast agents before viewing. Probably, with age, the bacterial cell is filled with fatty inclusions that do not perceive color and, as a result, cause the cell to be striated. The stage of "belted rods" precedes the stage of formation of bacteroids - irregularly shaped cells: thickened, branched, spherical, pear-shaped and flask-shaped. than rod-shaped bacterial cells found in nodule tissues.

Bacteroids contain more volutin granules and are characterized by a higher content of glycogen and fat than rod-shaped cells. Bacteroides grown in artificial nutrient media and formed in nodule tissues are physiologically of the same type. It is believed that bacteroids are forms of bacteria with an incomplete division process. With incomplete cell division of nodule bacteria, dichotomously branching forms of bacteroids arise. The number of bacteroids increases with the aging of the culture; their appearance is facilitated by the depletion of the nutrient medium, the accumulation of metabolic products, and the introduction of alkaloids into the medium.

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In old (two-month-old) cultures of nodule bacteria, using an electron microscope, it is possible to identify clearly defined spherical formations in many cells - arthrospores. Their number in cells varies from 1 to 5.

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Nodule bacteria of various types of leguminous plants grow at different rates on nutrient media. Fast-growing bacteria include rhizobia of peas, clover, alfalfa, fodder beans, vetch, lentils, chiny, sweet clover, fenugreek, beans, chickpeas, and bird-foot; to slow-growing - nodule bacteria of lupine, soybean, peanut, seradella, mung bean, cowpea, sainfoin, gorse. Fully formed colonies of fast-growing cultures can be obtained on the 3rd - 4th day of incubation, colonies of slow-growing cultures - on the 7th - 8th.

Fast-growing nodule bacteria are characterized by a peritrichous arrangement of flagella, while slow-growing bacteria are monotrichial.

In addition to flagella, filamentous and bead-like outgrowths are formed in nodule bacteria cells when grown on liquid media. Their length reaches 8-10 microns. They are usually located on the surface of the cell peritrichially, they contain from 4 to 10 or more per cell.

Nodule bacteria are microaerophiles (they develop with small amounts of oxygen in the environment), however, they prefer aerobic conditions.

Many types of nodule bacteria are able to synthesize B vitamins, as well as growth substances such as heteroauxin (beta-indoleacetic acid).

All nodule bacteria are approximately equally resistant to an alkaline reaction of the medium (pH = 8.0), but unequally sensitive to an acidic one.

Specificity, virulence, competitiveness and activity of nodule bacteria - the concept of the specificity of nodule bacteria is a collective one. It characterizes the ability of bacteria to form nodules in plants. If we talk about nodule bacteria in general, then for them the formation of nodules only in a group of leguminous plants is already specific in itself - they have selectivity for leguminous plants.

In the process of infection of the root system of leguminous plants with nodule bacteria, the virulence of microorganisms is of great importance. If specificity determines the spectrum of action of bacteria, then the virulence of nodule bacteria characterizes the activity of their action within this spectrum. Virulence refers to the ability of nodule bacteria to penetrate the root tissue, multiply there, and cause the formation of nodules.

An important role is played not only by the very ability to penetrate into the roots of the plant, but also by the speed of this penetration.

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In soil, in the presence of other strains, the more virulent strain will not always infect the plant first. In this case, one should take into account its competitive ability, which often masks the property of virulence in natural conditions.

An important property of nodule bacteria is their activity (efficiency), i.e. the ability in symbiosis with leguminous plants to assimilate molecular nitrogen and satisfy the needs of the host plant in it. Depending on the extent to which nodule bacteria contribute to an increase in the yield of legumes, they are usually divided into active (effective), inactive (ineffective) and inactive (ineffective).

Rice. 6 - A layer of granular substance on the surface of the root hair (1) during electron microscopic examination (according to P. Dart, F. Mercer, magnification X 30,000) and a slimy zoogley layer (2), in which clover root hairs are immersed, under a light microscope (zoom x 80)

Exposure to gamma rays makes it possible to obtain strains with enhanced efficiency. There are known cases of the emergence of highly active radiomutants of alfalfa nodule bacteria from an inactive strain. The use of ionizing radiation, which has a direct effect on the change in the genetic characteristics of the cell, in all likelihood, can be a promising technique in the selection of highly active strains of nodule bacteria.

Infection of a legume plant with nodule bacteria - to ensure the normal process of infection of the root system with nodule bacteria, it is necessary to have a fairly large number of viable bacterial cells in the root zone.

On the surface of the root there is a layer of mucous substance (matrix), which is formed regardless of the presence of bacteria in the rhizosphere. This layer is clearly visible when examined under a light-optical microscope. Nodule bacteria after inoculation usually rush to this layer and accumulate in it due to the stimulating effect of the root, which manifests itself even at a distance of up to 30 mm.

There are a number of hypotheses about the mechanism of penetration of nodule bacteria into the plant root. The most interesting of them are the following. The authors of one of the hypotheses state that nodule bacteria penetrate into the root through damage to the epidermal and cortical tissue (especially in the places where lateral roots branch off). This hypothesis was put forward on the basis of research by Bril (1888), who caused the formation of nodules in leguminous plants by piercing the roots with a needle previously immersed in a suspension of nodule bacteria. As a special case, such an implementation path is quite real. For example, in peanut nodules are predominantly located in the axils of root branches, which suggests the penetration of nodule bacteria into the root through gaps during the germination of lateral roots.

It is possible that nodule bacteria can penetrate into the root through the epidermal cells of young root tips. According to Prazhmovsky (1889), bacteria can penetrate the root only through the young cell wall (of root hairs or epidermal cells) and are completely incapable of overcoming the chemically altered or corky layer of the cortex. This may explain that nodules usually develop on young sections of the main root and emerging lateral roots.

Recently, the auxin hypothesis has received great popularity. The authors of this hypothesis believe that nodule bacteria penetrate into the root by stimulating the synthesis of p-indoleacetic acid (heteroauxin) from tryptophan, which is always present in plant root secretions. The presence of heteroauxin is associated with curvature of root hairs, which is usually observed when the root system is infected with nodule bacteria.

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The source of beta-indolylacetic acid at the time of infection of a plant, obviously, is not only plants that secrete tryptophan through the root system, which many types of bacteria, including root nodules, can convert into beta-indolylacetic acid. Nodule bacteria themselves, and possibly other types of soil microorganisms living in the root zone, can also participate in the synthesis of heteroauxin.

The action of heteroauxin is nonspecific and causes curvature of root hairs in various plant species, not just legumes. At the same time, nodule bacteria cause curvature of root hairs only in leguminous plants, while exhibiting rather significant selectivity. If the effect under consideration were determined only by 3-indolylacetic acid, then such specificity would not exist. In addition, the nature of changes in root hairs under the influence of nodule bacteria is somewhat different than under the influence of heteroauxin.

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nodule nitrogenous bacterium legume

Another hypothesis should be noted, according to which nodule bacteria enter the root during the formation of a finger-like protrusion on the surface of the root hair. An electron diffraction pattern of a root hair section confirming this hypothesis shows a root hair bent in the form of an umbrella handle, in the bend of which there is an accumulation of nodule bacteria. Nodule bacteria are as if drawn in (swallowed) by the root hair (similar to pinocytosis).

Rice. nine - Curvature of the root hairs of leguminous plants in the presence of nodule bacteria: 1.2 - stole, x 120; 3 - ultrathin section through the root hair. Increased X 10,000 (according to K. Salman and G. Fareus)

The process of introduction of nodule bacteria into the root tissue is the same in all types of leguminous plants and consists of two phases. In the first phase, infection of the root hairs occurs. In the second phase, the process of nodule formation proceeds intensively. The duration of the phases is different in different plant species: in Trifolium fragiferum the first phase lasts 6 days, in Trifolium nigrescens - 3 days. In some cases it is very difficult to detect the boundaries between phases. The most intensive introduction of nodule bacteria into root hairs occurs at the early stages of plant development. The second phase ends during the mass formation of nodules. Often, the penetration of nodule bacteria into root hairs continues even after the nodules have formed on the roots. This so-called excess or additional infection occurs because the infection of the hairs does not stop for a long time. In the later stages of infection, the nodules are usually placed lower along the root.

However, in most cases, the invading cell, actively multiplying, forms the so-called infectious threads (or infectious tji) and, already in the form of such threads, moves into the plant tissues.

The term "infection thread" arose on the basis of the study of the infection process in a light microscope. Starting with the work of Beijerinck, the infection thread began to be considered as a slimy hyphae-like mass with enclosed

According to the method of formation, nodules of leguminous plants are divided into two types: 1st type - nodules arise during the division of cells of the pericycle (root layer), usually located against the protoxylem (first in time for the formation of vessels) - endogenous type of formation of nodules; Type 2 - nodules originate from the root cortex as a result of the introduction of the pathogen into the parenchymal cells of the cortex and endoderm (the inner layer of the primary cortex) - an exogenous type of nodule formation.

Rice. 10 - Sections through the nodule tissue of leguminous plants: 1 - cross section through the nodule of peas. The beginning of the differentiation of vascular tissue in the nodule (according to G. Bond); d - longitudinal section through the nodule of the rank. Increased x90

In nature, the latter type predominates. The tissues of the central cylinder of the root take part only in the formation of the vascular system of nodules of both endogenous and exogenous types.

Rice. eleven - Intercellular infection threads in the form of a pocket (right) and a torch (left) with nodule bacteria. The arrow shows a bacterial cell preparing to enter the cytoplasm of a plant cell. Increased X 20,000

The formation of nodules of leguminous plants occurs during the period when the root still has a primary structure. It begins with the division of cortical cells located at a distance of 2-3 layers from the ends of the infectious threads. The layers of the cortex, penetrated by infectious threads, remain unchanged. At the same time, in seradella, division of cortical cells occurs directly under the infected root hair, while in peas, cell division is noted only in the penultimate layer of the cortex.

A secondary meristem appears. Soon, in the endoderm and pericycle, signs of incipient division appear, which in the former outer cells occurs mainly by tangential septa. This division finally extends to the common meristematic complex, the small cells of which are elongated, the vacuoles disappear, and the nucleus fills most of the cell. The so-called primary nodule is formed, in the plasma of cells of which nodule bacteria are absent, since at this stage they are still inside the infection threads. While the primary nodule is being formed, infection threads branch out many times and can pass either between cells - intercellularly, or through cells - intracellularly - and introduce bacteria.

Rice. 12 - Intracellular infectious threads with nodule bacteria: 1 - longitudinal section (according to D. Goodchild and F. Bergersen), 2 - cross section. Led away, x 35000

Intercellular infectious threads due to the active reproduction of nodule bacteria in them often acquire a bizarre shape - they are formed in the form of pockets (diverticula) or torches.

The process of movement of infection threads from cell to cell is not entirely clear. Apparently, infectious threads, as Canadian microbiologist D. Jordan believes, wander in the form of naked mucous strands in the intercellular spaces of plant tissue until, due to some still inexplicable reasons, they begin to invaginate into the cytoplasm of adjacent cells.

In some cases, the invagination of the infection thread occurs in one, in some cases - in each neighboring cell. Through these invaginated tubular cavities (diverticula), the contents of the thread enclosed in mucus flow. The most active growth of infectious threads usually occurs near the nucleus of the plant cell. The penetration of the thread is accompanied by the movement of the nucleus, which moves towards the site of infection, increases, changes shape and degenerates. A similar picture is observed in a fungal infection, when the nucleus often rushes towards the invading hyphae, is attracted to the damage as to the place of the greatest physiological activity, comes close to the thread, swells and collapses. Apparently, this is characteristic of the plant's response to infection.

In annual plants, infectious threads usually appear during the first period of infection of the root, in perennial plants - during a long period of development.

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Bacteria can be released from the infection thread at different times and in different ways. The exit of bacteria, as a rule, is a very long process, especially in perennial plants. Usually, the release of bacteria from the infection thread into the cytoplasm of the host plant is associated with internal pressure resulting from the intensive multiplication of bacteria in the thread and their excretion of mucus. Sometimes bacteria slip out of the thread in groups surrounded by the mucus of the infection thread. in the form of vesicles (vesicular formations). Since the vesicles do not have membranes, the exit of bacteria from them is very simple. Nodule bacteria can also enter plant cells singly from the intercellular spaces.

Nodule bacteria that have emerged from the infection thread continue to multiply in the host tissue. Their reproduction during this period occurs by constriction division. The bulk of bacteria multiply in the cytoplasm of the cell, and not in the infection thread. Infected cells give rise to future bacteroid tissue.

Filled with rapidly multiplying cells of nodule bacteria, plant cells begin to intensively divide. At the moment of mitotic division of infected cells, nodule bacteria can accumulate at two opposite poles of the mother cell and passively enter the daughter cells. Each of the uninfected cells is under a strong stimulating effect of nodule bacteria and, as a result, also divides. Thanks to this vigorous mitotic division of meristematic cells, nodule bacteria spread into nodule tissues and the volume of the bacteroid region increases.

The infected tissue, consisting of densely lying and actively dividing cells, first has the shape of a truncated cone. Subsequently, due to the gradual growth of this cone and the simultaneous division and development of meristematic cells, the nodule tissue grows, losing its cone shape.

Thus, the nodule increases first as a result of radial and tangential division of the core cells, and then due to an increase in their size and simultaneous division. After plant cells are completely filled with bacteria, mitosis stops. However, the cells continue to increase in size and are often highly elongated. Their size is several times larger than that of uninfected plant cells, which are located between them in the bacteroid zone of the nodule.

The connection of a young nodule with the root of a leguminous plant is carried out thanks to vascular-fibrous bundles. For the first time, vascular fibrous bundles were observed by M.S. Voronin (1866). The time of occurrence of the vascular system in the nodules of various types of leguminous plants is different. So, in soybean nodules, the beginning of the development of vascular bundles coincides with the moment of penetration of nodule bacteria into two layers of the cow's parenchyma. With the growth of the nodule, the conducting system grows, branches, and surrounds the bacteroid region.

In parallel with the process of differentiation of the vascular system, the nodule endoderm is formed from the outer layer of the primary nodule. Then the nodule is rounded, its peripheral cell layer is surrounded by nodule bark.

The root epidermis breaks, and the nodule continues to develop and increase in size.

Using a light microscope on longitudinal sections of mature nodules, 4 characteristic zones of tissue differentiation are usually clearly distinguished: cortex, meristem, bacteroid zone, and vascular system. All nodule tissues differentiate in an acropetal sequence as new cells are initiated by the meristem.

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Nodule bark - the shell of the nodule, which performs a protective function. The bark consists of several rows of uninfected parenchymal cells, the size and characteristics of which are different in different legumes. Most often, the cells of the cortex have an elongated shape and are larger in comparison with other cells of the nodule.

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In the bark of nodules of perennial woody species, cells with corky membranes containing resins, tannin, and tannins are often found.

The nodule meristem is located under the cells of the cortex and is a zone of intensively dividing also uninfected cells. The meristem of the nodule is characterized by densely spaced, without intercellular spaces, small thin-walled cells of irregular shape. Nodule meristem cells are similar to cells of other types of meristematic tissue (root top, stem top). Nodule meristem cells contain dense, finely granular cytoplasm with ribosomes, Golgi bodies, protoplastids, mitochondria, and other structures. There are small vacuoles. In the center of the cytoplasm is a large nucleus with a nuclear membrane, pores and a clearly defined nucleolus. The functions of meristematic cells are to form cells of the nodule cortex, bacteroid region and the vascular system. Depending on the location of the meristem, nodules have a variety of shapes: spherical (peas, beans, seradella, peanuts) or cylindrical (alfalfa, vetch, rank, acacia, clover). The meristem, located in separate areas along the periphery of the nodule, leads to the formation of muff-like nodules in lupine.

The nodule meristem functions for a long time, even during nodule necrosis, when they are already filled with lysing bacteroid mass and destroyed plant cells.

The bacteroid zone of a nodule occupies its central part and makes up from 16 to 50% of the total dry mass of nodules. In the first period of nodule formation, it is essentially a bacterial zone, since it is filled with bacterial cells that are in the bacterial rather than the bacteroid stage of development. Nevertheless, when it comes to the nodule tissue zone containing bacteria, it is customary to call it bacteroid.

The bacteroid region of the nodule consists mainly of cells infected with nodule bacteria and partly of uninfected cells adjacent to them, filled with pigments, tannins, and by autumn - with starch.

Rice. 16 - Form of nodules in leguminous plants: 1, 2 - peas; 3, 6 (right) - lupine; 4 - peanuts (according to E. Schwinghamer); 5 - ranks, clover, wiki, 6 (left) - alfalfa (according to J. Date); 7 - different types of wild leguminous plants (according to A.R. Werner, N.Ya. Gordienko)

In nodules formed by effective strains of nodule bacteria, the average relative volume of the bacteroid zone is higher than in nodules formed upon the introduction of ineffective strains.

In some cases, the volume of the bacteroid region reaches a maximum in early period life of the nodule and subsequently remains relatively constant. The bacteroid zone is penetrated by a dense network of infectious threads, and is surrounded by vascular fibrous bundles along the periphery.

The form of bacteroids in the nodules of different types of legumes can be varied. So, in wiki, rank and pea, they are two-branched or forked. For clover and sainfoin, the predominant form of bacteroids is spherical, pear-shaped, swollen, ovoid, and round for chickpeas. The shape of the bacteroides of the bean, seradella, bird-foot, and lupine is almost rod-shaped.

Bacteroides fill most of the plant cell, with the exception of the central zone of the nucleus and vacuoles. So, the percentage of bacteroids in the bacteroid zone stained in pink color nodule is 94.2 of the total number of nodule bacteria. Bacteroid cells are 3-5 times larger than bacterial cells.

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Bacteroides of nodule bacteria are of particular interest due to the fact that they are almost the only inhabitants of the nodules of leguminous plants during the period of intensive binding of atmospheric nitrogen by them. Some researchers consider bacteroids to be pathological degenerative forms and do not associate the process of nitrogen fixation with the bacteroid form of nodule bacteria. Most investigators find that bacteroids are the most viable and active forms of nodule bacteria and that legumes fix atmospheric nitrogen only with their participation.

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The vascular system of the nodule provides a link between the bacteria and the host plant. Nutrients and metabolic products are transported through the vascular bundles. The vascular system develops early and functions for a long time.

Fully formed vessels have a specific structure: they consist of xylem tracheids, phloem fibers, sieve tubes and accompanying cells.

Depending on the type of legume, the connection of the glomerulus is carried out through one or more vascular bundles. For example, in peas, there are two differentiated vascular nodes at the base of the nodule. Each of them usually branches dichotomously twice, and as a result, 8 bundles pass through the nodule from the place of the second dichotomous branching. Many plants have only one bunch, at the same time, in one Sesbania grandiflora nodule at the age of one year, they managed to count up to 126. Quite often, the vascular system of the nodule is separated on the outside from its bark by a layer of partially or completely suberized cells, called nodule endoderm, attached to the root endodermis. The nodule endoderm is the outer layer of uninfected bovine parenchyma, located between the nodule tissue and the root cortex.

In most plant species, nodules are formed according to the described type. Therefore, nodule formation is the result of complex phenomena starting outside the root. Following the initial phases of infection, the formation of a nodule is induced, then the spread of bacteria in the nodule tissue zone and nitrogen fixation occur.

All stages of development of nodule bacteria, according to the Czech microbiologist V. Kas (1928), can be traced on sections of nodules. So, in the upper part of the nodule, for example, alfalfa contains mainly small dividing rod-shaped cells, a small amount of young bacteroids, the number of which increases gradually as the nodule develops. In the middle, pink-colored part of the nodule, predominantly bacteroid cells and less often small rod-shaped cells are found. At the base of the nodule in the early stages of vegetation of the host plant, the bacteroids are the same as in its middle part, but by the end of the growing season they are more swollen and degenerate earlier.

The timing of the appearance of the first visible nodules on the roots of various types of leguminous plants is different. Their appearance in most legumes most often occurs during the development of the first true leaves. Thus, the formation of the first nodules of alfalfa is observed between the 4th and 5th days after germination, and on the 7th - 8th day this process occurs in all plants. The nodules of sickle alfalfa appear after 10 days.

During the period of functioning, the nodules are usually dense. Nodules formed by active cultures of bacteria are whitish in color at a young age. By the time of manifestation of optimal activity, they become pink. Nodules that have arisen during infection with inactive bacterial cultures are greenish in color. Often, their structure practically does not differ from the structure of nodules formed with the participation of active strains of nodule bacteria, but they are destroyed prematurely.

In some cases, the structure of nodules formed by inactive bacteria deviates from the norm. This is expressed in the disorganization of the nodule tissue, which usually loses its clearly defined zonal differentiation.

The pink color is determined by the presence of a pigment in the nodules, which is similar in chemical composition to blood hemoglobin. In this regard, the pigment is called leghemoglobin (legoglobin) - Leguminosae hemoglobin. Legoglobin is found only in those nodule cells that contain bacteroids. It is localized in the space between the bacteroids and the membrane surrounding them.

Its amount ranges from 1 to 3 mg per 1 g of nodule, depending on the type of leguminous plant.

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In annual leguminous plants, by the end of the growing season, when the process of nitrogen fixation ends, the red pigment turns into green. The color change begins at the base of the nodule, later its top turns green. In perennial leguminous plants, greening of nodules does not occur or it is observed only at the base of the nodule. In different types of leguminous plants, the transition of red pigment to green occurs with varying degrees of intensity and at different rates.

Nodules of annual plants function for a relatively short time. In most legumes, nodule necrosis begins during the flowering period of the host plant and usually proceeds from the center to the periphery of the nodule. One of the first signs of destruction is the formation of a layer of cells with powerful walls at the base of the nodule. This layer of cells, located perpendicular to the main root vessel, separates it from the nodule and delays the exchange of nutrients between the host plant and nodule tissues.

Numerous vacuoles appear in the cells of the degenerating tissue of the nodule, the nuclei lose their ability to stain, some of the nodule bacteria cells lyse, and some migrate into the environment in the form of small coccoid arthrospore cells.

Old nodules are dark, flabby, soft. When cut, watery mucus protrudes from them. The process of nodule destruction, which begins with corking of the cells of the vascular system, is facilitated by a decrease in the photosynthetic activity of the plant, dryness or excessive humidity of the environment.

In a destroyed, mucilaginous nodule, protozoa, fungi, bacilli, and small rod-shaped nodule bacteria are often found.

The state of the host plant affects the duration of the functioning of the nodule. Thus, according to F. F. Yukhimchuk (1957), by castrating or removing lupine flowers, it is possible to prolong the period of its vegetation and, at the same time, the time of active activity of nodule bacteria.

Nodules of perennial plants, unlike annual nodules, can function for many years. So, for example, caragana has perennial nodules, in which the process of cell aging occurs simultaneously with the formation of new ones. In wisteria (Chinese wisteria), perennial nodules also function, forming spherical swellings on the roots of the host. By the end of the growing season, the bacteroid tissue of perennial nodules degrades, but the entire nodule does not die. The next year it starts functioning again.

Factors that determine the symbiotic relationship of nodule bacteria with leguminous plants - for a symbiosis that provides good development plants, a certain set of environmental conditions is necessary. If the environmental conditions are unfavorable, then, even despite the high virulence, competitive ability and activity of the microsymbiont, the effectiveness of the symbiosis will be low.

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For the development of nodules, the optimal moisture content is 60-70% of the total moisture capacity of the soil. The minimum soil moisture at which the development of nodule bacteria in the soil is still possible is approximately equal to 16% of the total moisture capacity. When humidity is below this limit, nodule bacteria usually no longer multiply, but nevertheless they do not die and can remain in an inactive state for a long time. The lack of moisture also leads to the death of already formed nodules.

Excess moisture, as well as its lack, is also unfavorable for symbiosis - due to a decrease in the degree of aeration in the root zone, the supply of oxygen to the root system of the plant worsens. Insufficient aeration also negatively affects nodule bacteria living in the soil, which, as you know, multiply better when oxygen is available. Nevertheless, high aeration in the root zone leads to the fact that molecular nitrogen reducers begin to bind oxygen, reducing the degree of nitrogen fixation of nodules.

The temperature factor plays an important role in the relationship between nodule bacteria and leguminous plants. The temperature characteristics of different types of leguminous plants are different. Also, different strains of nodule bacteria have their own specific temperature optimums for development and active nitrogen fixation. It should be noted that the optimal temperatures for the development of leguminous plants, the formation of nodules and nitrogen fixation do not coincide. So, under natural conditions, the formation of nodules can be observed at temperatures slightly above 0°C, nitrogen fixation practically does not occur under such conditions. Perhaps only arctic symbiotic leguminous plants fix nitrogen at very high temperatures. low temperatures. Usually, this process occurs only at 10°C and above. The maximum nitrogen fixation of a number of leguminous plants is observed at 20-25°C. Temperatures above 30°C adversely affect the process of nitrogen accumulation.

The soil reaction has a great influence on the vital activity of nodule bacteria and the formation of nodules. For different species and even strains of nodule bacteria, the pH value of the habitat is somewhat different. For example, clover nodule bacteria are more resistant to low pH values than alfalfa nodule bacteria. Obviously, the adaptation of microorganisms to the environment also affects here. Clover grows in more acidic soils than alfalfa. Soil reaction as an ecological factor affects the activity and virulence of nodule bacteria. The most active strains tend to be easier to isolate from soils with neutral pH values. In acidic soils, inactive and weakly virulent strains are more common. An acidic environment (pH 4.0 - 4.5) has a direct effect on plants, in particular, disrupting the synthetic processes of plant metabolism and the normal development of root hairs. In an acidic environment in inoculated plants, the period of functioning of the bacteroid tissue is sharply reduced, which leads to a decrease in the degree of nitrogen fixation.

In acidic soils, as noted by A.V. Petersburg, salts of aluminum and manganese pass into the soil solution, adversely affecting the development of the root system of plants and the process of nitrogen assimilation, and the content of assimilable forms of phosphorus, calcium, molybdenum and carbon dioxide also decreases. Unfavorable soil reaction is best eliminated by liming.

For nodule bacteria, the host tissue is such a nutrient medium that can satisfy even the most demanding strain due to the content of all types of nutrients in the tissue. Nevertheless, after the introduction of nodule bacteria into the tissue of the host plant, their development is determined not only internal processes, but also depends to a large extent on the action external factors that affect the entire course of the infectious process. The content or absence of a nutrient in environment can be a defining moment for the manifestation of symbiotic nitrogen fixation.

The degree of supply of legume plants with available forms of mineral nitrogen compounds determines the effectiveness of symbiosis. Based on numerous laboratory and vegetative experiments, it is known that the more nitrogen-containing compounds in the environment, the more difficult it is for bacteria to penetrate into the root.

Of great importance in activating the absorption of nitrogen by legumes is phosphorus nutrition. At a low phosphorus content in the medium, bacteria penetrate into the root, but nodules are not formed. Leguminous plants have some peculiarities in the exchange of phosphorus-containing compounds. Legume seeds are high in phosphorus. Reserve phosphorus during seed germination is not used in the same way as in other crops - relatively evenly for the formation of all organs, but to a greater extent concentrated in the roots. Therefore, in the early stages of development, leguminous plants, unlike cereals, satisfy their needs for phosphorus to a greater extent at the expense of cotyledons, and not soil reserves. The larger the seeds, the less legumes depend on soil phosphorus. However, with a symbiotic mode of existence, the need for phosphorus in leguminous plants is higher than with an autotrophic one. Therefore, with a lack of phosphorus in the medium of inoculated plants, the supply of plants with nitrogen worsens.

Symbiotic nitrogen fixation also requires magnesium, sulfur and iron. With a lack of magnesium, the reproduction of nodule bacteria is inhibited, their vital activity decreases, and symbiotic nitrogen fixation is suppressed. Sulfur and iron also have a beneficial effect on the formation of nodules and the process of nitrogen fixation, in particular, playing an undoubted role in the synthesis of leghemoglobin.

Of the trace elements, we especially note the role of molybdenum and boron. With a lack of molybdenum, nodules are poorly formed, the synthesis of free amino acids is disturbed in them, and the synthesis of leghemoglobin is suppressed. Molybdenum, together with other elements with variable valence (Fe, Co, Cu) serves as an intermediary in the transfer of electrons in redox enzymatic reactions. With a boron deficiency, vascular bundles are not formed in the nodules, and as a result, the development of bacteroid tissue is disrupted.

The formation of nodules in legumes is greatly influenced by carbohydrate metabolism plants, determined by a number of factors: photosynthesis, the presence in the environment carbon dioxide, physiological characteristics of plants. Improving carbohydrate nutrition has a positive effect on the inoculation process and nitrogen accumulation. From a practical point of view, the use of straw and straw fresh manure for fertilizing leguminous plants as a source of carbohydrates is of great interest. But in the first year after the introduction of straw into the soil, toxic substances accumulate during its decomposition. It should be noted that not all types of leguminous plants are sensitive to toxic decomposition products of straw; peas, for example, do not react to them.

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