The role of nodule bacteria is: The role of nodule bacteria

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

U modern species leguminous plants, nodules are found on the roots of many representatives 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 detected in only approximately 1,300 species (243 genera). This primarily includes plant species used in agriculture (more than 200).

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

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

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

20 years later, the Dutch scientist Beijerin isolated bacteria from the nodules of peas, vetch, china, beans, seradella and commonweed and studied their properties, testing their ability to infect plants and cause the formation of nodules. He named these microorganisms Bacillus radicicola. Since the genus Bacillus includes bacteria that form spores, 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 literature today.

To designate a species of nodule bacteria, it is customary to add to the generic name Rhizobium a term 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 - nodule bacteria of clover, Rhizobium lupini - nodule bacteria of lupine, etc. In cases where nodule bacteria are capable of forming nodules on the roots of different types of leguminous plants, i.e., causing so-called cross-infection, the species name is as if collective - it reflects precisely this “cross-infecting” ability. For example, Rhizobium leguminosarum - nodule bacteria of peas (Pisum), lentils (Lens), and chin (Lathyrus).

Morphology and physiology of nodule bacteria. Nodule bacteria are characterized by an amazing variety of forms - polymorphism. Many researchers paid attention to this when studying nodule bacteria in pure culture in laboratory conditions and in soil. Nodule bacteria can be rod-shaped or oval. Among these bacteria there are also filterable forms, L-forms, coccoid immobile and motile 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, and reproduce by division. In the rod-shaped cells of clover nodule bacteria, division by lacing is observed. With age, rod-shaped cells may progress 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 turn into the state of so-called girdled rods. They received this name due to the alternation of dense and loose sections of protoplasm in the cells. The banding of cells is clearly visible when viewed under a light microscope after treatment of cells with aniline dyes. Dense areas of protoplasm (belts) 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 (Fig. 143, 1). The belts can be located in the middle of the cage or at the ends. The girdling of cells is also visible on electron diffraction patterns if the preparation is not treated with contrasting substances before viewing (Fig. 143, 3). Probably, with age, the bacterial cell becomes filled with fatty inclusions that do not perceive color and, as a result, cause the cell to become striated. The stage of “girdled rods” precedes the stage of formation of bacteroids - cells irregular shape: thickened, branched, spherical, pear-shaped and flask-shaped (Fig. 144). The term “bacteroids” was introduced into the literature by J. Brunkhorst in 1885, applying it to formations of unusual shape, much larger than rod-shaped bacterial cells, found in the tissues of nodules.

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

In old (two-month) 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 different types of legumes grow at different rates. The fast-growing ones include nodule bacteria of peas, clover, alfalfa, broad beans, vetch, lentils, china, sweet clover, fenugreek, beans, chickpeas, sweet grass; slow-growing - nodule bacteria of lupine, soybean, peanut, seradella, mung bean, cowpea, sainfoin, gorse. Fully formed colonies fast growing crops can be obtained on the 3rd - 4th day of incubation, slow-growing colonies - on the 7th - 8th.

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

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

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Colonies of fast-growing nodule bacteria are the color of baked milk, often translucent, slimy, with smooth edges, moderately convex, and grow over time 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 in small amounts of oxygen in the environment), however, they prefer aerobic conditions.

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

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

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

All nodule bacteria are approximately equally resistant to an alkaline reaction of the environment (pH = 8.0), but are not equally sensitive to an acidic environment.

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 in itself specific - 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 capable of infecting only a certain, sometimes large, sometimes smaller, group of leguminous plants, and in this sense, the specificity of nodule bacteria is their 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 non-alkaloid varieties of lupine). With broad specificity, nodule bacteria of peas can infect pea, china, and bean plants, and nodule bacteria of china and beans can infect pea plants, i.e., they are all characterized by the ability of “cross-infection.” 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 a different adaptability of nodule bacteria to plants within the cross-infection group. Thus, nodule bacteria in alfalfa can form nodules in sweet clover. But nevertheless, they are more adapted to alfalfa, and sweet clover bacteria - to sweet clover.

In the process of infection of the root system of leguminous plants by nodule bacteria, it is of great importance 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 root tissue, multiply there and cause the formation of nodules.

An important role is played not only by the ability to penetrate into the roots of a 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 ensures more vigorous infection of the roots compared to other strains, resulting 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, it should be taken into account competitive ability, which often masks the property of virulence in natural conditions.

It is necessary that virulent strains also have competitiveness, that is, 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 it forms 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, in symbiosis with leguminous plants, to assimilate molecular nitrogen and satisfy the needs of the host plant. Depending on the extent to which nodule bacteria contribute to increasing the yield of legumes (Fig. 146), they are usually divided into active (effective), inactive (ineffective) and inactive (ineffective).

A strain of bacteria that is inactive for one host plant in symbiosis with another type of legume 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 constant property. Often in laboratory practice, a loss of activity is observed 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 and amino acids. One of the reasons for the loss of activity of nodule bacteria may be the influence of phage. By passaging, i.e., repeatedly passing bacteria through a host plant (adaptation to a specific 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 changing 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 by nodule bacteria, it is necessary to have sufficient large quantity viable bacterial cells in the root zone. Researchers have different opinions regarding the number of cells required to ensure the inoculation process. Thus, according to the American scientist O. Allen (1966), for inoculation of small-seeded plants, 500-1000 cells are required, for inoculation of large-seeded plants - at least 70,000 cells per 1 seed. According to the Australian researcher J. Vincent (1966), at the time of inoculation, each seed should contain at least several hundred viable and active cells of nodule bacteria. There is evidence that single cells can invade root tissue.

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

In the rhizosphere of a legume 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 forms regardless of the presence of bacteria in the rhizosphere. This layer is clearly visible when examined under a light optical microscope (Fig. 147). After inoculation, nodule bacteria 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.

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

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

The hypothesis about the penetration of nodule bacteria into root tissue through root hairs is interesting and not without foundation. The route of passage of nodule bacteria through root hairs is recognized by most researchers.

The assumption 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 at 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 that the cells of nodule bacteria become smaller in the rhizosphere of legume plants confirm this position.

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

IN Lately The auxin hypothesis has gained 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 root secretions of plants. The presence of heteroauxin is associated with the curvature of root hairs, which is usually observed when the root system is infected by nodule bacteria ( Fig. 150).

The source of β-indoleacetic acid at the time of plant infection is obviously not only plants that secrete tryptophan through the root system, which many types of bacteria, including nodule bacteria, can convert into β-indoleacetic acid. The 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 different plant species, not just legumes. At the same time, nodule bacteria cause curvature of root hairs only in leguminous plants, showing quite significant selectivity. If the effect in question were determined only by β-indoleacetic 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.

It should also be noted that in some cases, non-curved root hairs become infected. Observations show that in alfalfa and peas, 60-70% of root hairs bend and curl, and in clover - about 50%. In some types of clover, this reaction is observed in no more than 1/4 of the infected hairs. In the curvature reaction, the condition of the root hair is obviously of great importance. Growing root hairs are most sensitive to the action of substances produced by bacteria.

It is known that nodule bacteria 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 plants to synthesize the enzyme polygalacturonase. This enzyme, by destroying pectin substances, affects the membrane of root hairs, making it more plastic and permeable. Polygalacturonase is always present in small quantities 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 the root thanks to companion bacteria that produce pectinolytic enzymes. This hypothesis was put forward based on the following facts. When microscopying root hairs, many researchers noted the presence of a light spot around which nodule bacteria accumulate. This spot may be a sign of the beginning of maceration (destruction) of tissue by protopectinase, similar to 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 the root.

Another hypothesis should be noted, according to which nodule bacteria enter the root during the formation of a finger-like invagination of the surface of the root hair. The electron diffraction pattern of a section of a root hair, which confirms this hypothesis (Fig. 150, 3), shows a root hair curved in the shape of an umbrella handle, in the bend of which there is an accumulation of nodule bacteria. Nodule bacteria seem to be drawn in (swallowed) by the root hair (similar to pinocytosis).

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

The process of introduction of nodule bacteria into root tissue is the same for 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 occurs intensively. The duration of the phases varies 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 boundaries between phases. The most intensive penetration of nodule bacteria into root hairs occurs in the early stages of plant development. The second phase ends during the period of mass formation of nodules. Often, the introduction 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. At later stages of infection, the nodules are usually located lower along the root.

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

Having penetrated the root (through the root hair, epidermal cell, and sites of root damage), nodule bacteria then move into the plant root tissue. Bacteria most easily pass through intercellular spaces.

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

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

The term “infection thread” arose from studying the infection process under a light microscope. Beginning with the work of Beijerinck, the infection thread began to be viewed as a mucous hyphae-like mass with replicating bacteria enclosed within it.

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 bacteria penetrate the root.

The number of infected root hairs varies significantly among individual plants. Typically, infection threads appear in deformed, twisted root hairs. However, there are indications that similar threads are sometimes found in straight hairs. Most 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 several have common threads of infection, giving rise to one nodule (Fig. 151).

The percentage of infected root hairs in the total number of deformed ones 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 infection threads there are many (up to 80%) so-called abortive threads that have stopped developing. The rate of advancement of normally developing infection threads in a plant is 5 - 8 microns per hour. At this speed, the infection thread can travel through a root hair 100-200 microns long in one day.

Morphological and anatomical characteristics of nodules in their ontogenesis.

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

1st type - nodules arise during the division of cells of the pericycle (root layer), usually located opposite the protoxylem (the 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 a pathogen into the parenchyma cells of the cortex and endoderm (inner layer of the primary cortex) - 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 the zkzo- and endotypes, their development process 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 individual features similarities in their origin. 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 of the development of lateral roots and nodules emphasize profound differences in the type of their formation. Lateral roots arise in the pericycle. From the very first moments of development, they are associated with the central cylinder of the main root, from which the central cylinders of the lateral roots branch, and they always arise against the ray of the primary wood. The formation of a nodule, unlike a 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 to the vessels of the root through the tracheid zone and have their own endoderm (Fig. 152).

The difference in the nature of the emergence of nodules 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 the nodules become visible very quickly after infection of the root by 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 core cells located at a distance of 2-3 layers from the ends of the infection threads. The layers of the cortex, penetrated by infectious threads, remain unchanged. At the same time, in Seradella, the division of bark cells occurs directly under the infected root hair, and in pea, cell division is observed only in the penultimate layer of the bark.

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

The divided cells of the cortex change: the nuclei become rounded and increase in size, the nucleoli especially increase. After mitosis, the nuclei disperse and, without taking their original shape, begin to divide again.

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

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Intercellular infection 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 (see Fig. 154).

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

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

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

Bacteria can be released from the infection thread in different time and in different ways. The release of bacteria is usually a very long process, especially in perennial plants. Typically, the release of bacteria from the infection thread into the cytoplasm of the host plant is associated with internal pressure resulting from the intensive reproduction 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 infectious thread, in the form of vesicles (bubble-like formations) (Fig. 157). Since the vesicles do not have membranes, the exit of bacteria from them is very easy. Nodule bacteria can also enter plant cells individually from intercellular spaces (Fig. 156).

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Nodule bacteria emerging from the infection thread continue to multiply in the host tissue. Their reproduction during this period occurs by division by constriction (Fig. 158). 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.

Plant cells filled with rapidly multiplying cells of nodule bacteria begin to rapidly divide. At the time 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 uncharged cells is under the strong stimulating influence of nodule bacteria and, as a result, also divides. Thanks to this vigorous mitotic division of meristematic cells, nodule bacteria spread into the nodule tissue and increase the volume of the bacteroid region.

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 conical 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. Once the plant cells are completely filled with bacteria, mitosis stops. However, the cells continue to increase in size and often become very 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 legume 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 appearance of the vascular system in the nodules of different types of legume plants is different. Thus, 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 bark parenchyma. As the nodule grows, the conducting system grows, branches, and surrounds the bacteroid region.

In parallel with the process of differentiation of the vascular system, the formation of nodule endoderm from the outer layer of the primary nodule occurs. Then the nodule becomes rounded, its peripheral cell layer is surrounded by a nodule cortex.

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

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

Nodule bark- the shell of the nodule, which performs a protective function. The bark consists of several rows of uninfected parenchyma cells, the size and characteristics of which vary in different legumes. Most often, cortical cells have elongated shape and larger compared to other nodule cells.

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

Nodule meristem located under the cells of the cortex and represents a zone of intensively dividing also uninfected cells. The nodule meristem is characterized by densely spaced, small thin-walled cells of irregular shape, without intercellular spaces. The cells of the nodule meristem are similar to the cells of other types of meristematic tissue (root apex, stem apex). The cells of the nodule meristem contain dense, finely granular cytoplasm with ribosomes, Golgi bodies, protoplastids, mitochondria and other structures. Small vacuoles are found. In the center of the cytoplasm there is a large nucleus with a nuclear membrane, pores and a clearly defined nucleolus. The functions of meristematic cells include the formation of cells of the nodule cortex, bacteroid region and vascular system. Depending on the location of the meristem, the nodules have a variety of shapes: spherical (peas, beans, seradella, peanuts) or cylindrical (alfalfa, vetch, china, acacia, clover) (Fig. 159). The meristem, located in separate sections along the periphery of the nodule, leads to the formation of muff-shaped nodules in lupine.

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

Bacteroid zone the nodule occupies its central part and makes up from 16 to 50% of the total dry mass of the nodules. During the first period of nodule formation, it is essentially a bacterial zone (Fig. 160), since it is filled with bacterial cells that are in the bacterial rather than bacteroid stage of development. However, it is accepted when we're talking about about the zone of nodule tissue containing bacteria, call it bacteroid.

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

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

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

The shape of bacteroids in the nodules of different types of legumes can be varied (Table 44). So, in vetch, china and peas they are two-branched or forked. For clover and sainfoin, the predominant shape of bacteroids is spherical, pear-shaped, swollen, ovoid, for chickpeas it is round. The shape of the bacteroides of beans, seradella, commonweed 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. Thus, the percentage of bacteroids in the bacteroid zone of a pink-colored nodule is 94.2 to the total number of nodule bacteria. Bacteroides cells are 3-5 times larger than bacterial cells (Fig. 161, 1, 2).

Bacteroides of nodule bacteria are of particular interest due to the fact that they are almost the only inhabitants of legume plant nodules during the period of intensive binding of atmospheric nitrogen. Some researchers consider bacteroides to be pathological degenerative forms and do not associate the process of nitrogen fixation with the bacteroid form of nodule bacteria. Most researchers find that bacteroides are the most viable and active forms of nodule bacteria and that the fixation of atmospheric nitrogen by leguminous plants occurs only with their participation (Fig. 162).

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

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

Depending on the type of legume, the nodule is connected 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 bundle, while in one Sesbania grandiflora nodule at the age of one year they could be counted up to 126. Quite often, the vascular system of a nodule is separated from the outer side from its bark by a layer of partially or completely suberized cells, called nodule endoderm, connected to the endodermis of the root. The nodule endoderm is the outer layer of uninfected cortical parenchyma located between the nodule tissue and the root cortex.

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

All stages of development of nodule bacteria, according to the Czech microbiologist V. Kas (1928), can be traced on sections of nodules. Thus, in the upper part of a nodule, for example, alfalfa, there are mainly small dividing rod-shaped cells, in 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 the growing season of the host plant, the bacteroids are the same as in its middle part, and 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 different types of leguminous plants is different (M. V. Fedorov, 1952). 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. Nodules of sickle alfalfa appear after 10 days.

During the period of functioning, the nodules are usually dense. Nodules formed by active bacterial cultures are whitish in color when young. By the time optimal activity occurs, they turn pink. Nodules that arise during infection with inactive bacterial cultures are greenish in color. Often their structure is practically no different 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 in the nodules of a pigment that is similar in chemical composition to blood hemoglobin. In connection with this, the pigment is called leghemoglobin (legoglobin) - Leguminosae hemoglobin. Leghemoglobin is contained only in those nodule cells that contain bacteroids. It is localized in the space between the bacteroids and the surrounding membrane.

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

In annual legumes, towards the end growing season When the process of nitrogen fixation ends, the red pigment turns 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 different degrees of intensity and at different speeds.

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 in the direction 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 thick walls at the base of the nodule. This layer of cells, located perpendicular to the main vessel of the root, disconnects it from the nodule and delays the exchange of nutrients between the host plant and the nodule tissues.

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

The process of arthrospore formation in the tissue of a lysing nodule is shown in Figures 163-165. The infection threads also stop functioning during this period (Fig. 166). The host cells lose turgor and are compressed by those neighboring cells that still have it.

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Old nodules are dark, flabby, soft. When cut, watery mucus emerges from them. The process of destruction of the nodule, which begins with suberization 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, slimed nodule, protozoa, fungi, bacilli and small rod-shaped nodule bacteria are often found.

The state of the host plant affects the duration of the nodule's functioning. Thus, according to F.F. Yukhimchuk (1957), by castrating or removing lupine flowers, you can extend its growing season and, at the same time, the time active work nodule bacteria.

Nodules of perennial plants, unlike nodules of annual plants, can function for many years. 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 following year it begins to function again.

Factors determining the symbiotic relationship of nodule bacteria with leguminous plants. For symbiosis to ensure good plant development, a certain set of environmental conditions is necessary. If 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.

For the development of nodules, the optimal humidity 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. At humidity below this limit, nodule bacteria usually no longer reproduce, but nevertheless they do not die and can remain in an inactive state for a long time. Lack of moisture also leads to the death of already formed nodules.

Often in areas with insufficient moisture, many legume plants develop without forming nodules.

Since nodule bacteria multiply in the absence of moisture, in the event of a dry spring, inoculated (artificially infected) seeds must be introduced deeper into the soil. For example, in Australia, seeds with nodule bacteria applied to them are deeply embedded in the soil. Interestingly, nodule bacteria from soils in arid climates tolerate drought more resistantly than bacteria from soils from humid climates. This demonstrates their ecological adaptability.

Excessive humidity, like 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 deteriorates. Insufficient aeration also negatively affects the nodule bacteria living in the soil, which, as is known, multiply better with access to oxygen. Nevertheless, high aeration in the root zone leads to the fact that reducing agents of molecular nitrogen begin to bind oxygen, reducing the degree of nitrogen fixation of nodules.

Temperature plays an important role in the relationship between nodule bacteria and leguminous plants. The temperature characteristics of different types of legume 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 legume plants, nodule formation and nitrogen fixation do not coincide. Thus, 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 symbiosis legumes fix nitrogen at very low temperatures. Typically, this process occurs only at 10 °C and above. Maximum nitrogen fixation of a number of leguminous plants is observed at 20-25 °C. Temperatures above 30 °C negatively affect the process of nitrogen accumulation.

Ecological adaptation to the temperature factor in nodule bacteria is much less than in many typical saprophytic forms. According to E.N. Mishustin (1970), this is explained by the fact that the natural habitat of nodule bacteria is plant tissue, where temperature conditions are regulated by the host plant.

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 their environment also plays a role here. Clover grows in more acidic soils than alfalfa. How does the soil react? environmental factor influences the activity and virulence of nodule bacteria. The most active strains are usually 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, the life of the bacteroid tissue in inoculated plants is sharply reduced, which leads to a decrease in the degree of nitrogen fixation.

In acidic soils, as noted by A.V. Petersburgsky, aluminum and manganese salts pass into the soil solution, which adversely affect the development of the root system of plants and the process of nitrogen absorption, and the content of digestible forms of phosphorus, calcium, molybdenum and carbon dioxide decreases. Unfavorable soil reactions are best eliminated by liming.

The extent of symbiotic nitrogen fixation is determined to a large extent by the nutritional conditions of the host plant, and not by the nodule bacteria. Nodule bacteria, as endotrophic plant symbionts, depend mainly on the plant to obtain carbon-containing substances and mineral nutrients.

For nodule bacteria, the host tissue provides a nutrient medium that can satisfy even the most demanding strain due to the content of all types of nutrients in the tissue. However, after the introduction of nodule bacteria into the tissue of the host plant, their development is determined not only by internal processes, but also largely depends on the action external factors, influencing the entire course of the infectious process. The content or absence of a particular nutrient in the environment may be the determining factor for the manifestation of symbiotic nitrogen fixation.

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

Agricultural practice requires an unambiguous solution to the problem - is it more expedient to fertilize legumes with nitrogen, or are those researchers right who claim that mineral nitrogen suppresses the symbiotic nitrogen fixation of legumes and therefore it is more economically profitable not to fertilize such plants with nitrogen. At the Department of Agronomic and Biological Chemistry of the Moscow Agricultural Academy. K. A. Timiryazev conducted experiments, the results of which made it possible to obtain a picture of the behavior of symbionts under conditions of vegetation and field experiments when plants were provided with different doses of nitrogen in the environment. It has been established that increasing the content of soluble nitrogen-containing compounds in the environment under field conditions under optimal plant growth conditions does not prevent their symbiosis with nodule bacteria. A decrease in the proportion of atmospheric nitrogen absorbed by plants with an increased supply of mineral nitrogen is only relative. The absolute amount of nitrogen absorbed by bacteria from the atmosphere practically does not decrease, and even often increases compared to plants grown in the presence of nodule bacteria, but without adding nitrogen to the soil.

Phosphorus nutrition is of great importance in activating the absorption of nitrogen by legumes. With a low phosphorus content in the medium, bacteria penetrate into the root, but nodules do not form. Leguminous plants have some peculiarities in the metabolism of phosphorus-containing compounds. Legume seeds have a high phosphorus content. During seed germination, reserve phosphorus is used not 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 early dates During development, legume plants, unlike cereals, largely satisfy their phosphorus needs from cotyledons rather than from soil reserves. The larger the seeds, the less legume plants depend on soil phosphorus. However, with a symbiotic mode of existence, the need of legume plants for phosphorus is higher than with an autotrophic one. Therefore, if there is a lack of phosphorus in the environment, the nitrogen supply of inoculated plants deteriorates.

Legumes are known to yield significantly more potassium than other crops. Therefore, potassium and especially phosphorus-potassium fertilizers significantly increase the productivity of nitrogen fixation by legumes.

The positive effect of potassium on the formation of nodules and the intensity of nitrogen fixation is largely due to the physiological role of potassium in the carbohydrate metabolism of the plant.

Calcium is needed not only to eliminate excessive soil acidity. It plays a specific role in the development of nodule bacteria and ensuring normal symbiosis of bacteria with the host plant. The calcium requirement of nodule bacteria can be partially compensated for by strontium. Interestingly, nodule bacteria of tropical crops growing on acidic lateritic soils do not require calcium. This again demonstrates the ecological adaptation of nodule bacteria, since tropical soils contain very small amounts of calcium.

Magnesium, sulfur and iron are also required for symbiotic nitrogen fixation. With a lack of magnesium, the reproduction of nodule bacteria is inhibited, their vital activity is reduced, 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 microelements, 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 disrupted 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 enzyme reactions. With boron deficiency, vascular bundles do not form in the nodules, and as a result, the development of bacteroid tissue is disrupted.

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

Biological factors have a certain significance in the symbiosis of nodule bacteria and leguminous plants.

Much attention is paid to the influence of rhizosphere microflora on nodule bacteria, which can be both stimulatory and antagonistic in nature, depending on the composition of rhizosphere microorganisms.

Many works are devoted to the study of phages of nodule bacteria. Most phages are capable of lysing various types of bacteria; some are specialized only in relation to certain species or even strains of nodule bacteria. Phages can prevent the penetration of bacteria into the root and cause cell lysis in the nodule tissue. Phages cause great damage by lysing preparations of nodule bacteria in factories that produce nitragine.

Among the various types of insects that harm nodule bacteria, the striped nodule weevil stands out especially, the larvae of which destroy nodules on the roots of many species of legumes (mainly annuals). The bristly nodule weevil is also widespread.

In early spring, female nodule weevils lay 10 to 100 eggs. After 10-15 days, small (up to 5.5 mm), worm-shaped, bent, white larvae with a light brown head develop from the eggs, feeding mainly on nodules and root hairs. Newly hatched larvae penetrate the nodule and feed on its contents. More adult larvae destroy the nodules from the outside. One larva destroys 2-6 nodules in 30-40 days. They cause especially great harm in dry and hot weather when plant development slows down.

The nodules of alfalfa and some other types of leguminous plants are also damaged by the large alfalfa weevil.

Female beetles lay up to 400 eggs, from which legless, arched, yellowish-white larvae with a brown head and covered with brown bristles develop. Their length is 10-14 mm. The development cycle of the large alfalfa weevil takes place over two years.

In steppe regions, a steppe nematode was found on the roots of alfalfa, clover and soybeans. Before laying eggs, females penetrate into the root, where they lay from 12 to 20 eggs. In the roots, the larvae go through three larval stages of development, disrupting the functions of the root and nodules.

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-climatic conditions and the ability to lead a symbiotic and saprophytic way of life.

By schematizing the currently available data on the distribution of nodule bacteria in nature, the following generalizations can be made.

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

In nature and agricultural practice, cross-infection of different types of leguminous plants often leads to the appearance of nodules on the roots that are not sufficiently active in fixing molecular nitrogen. This usually depends on the absence of the corresponding species 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 bacteria of cross groups or develop without nodules.

Nodules in plants other than legumes.

Root nodules or nodule-like formations are widespread on the roots of not only leguminous plants. They are found in gymnosperms and angiosperms and dicotyledonous plants.

There are up to 200 types various plants that bind nitrogen in symbiosis with microorganisms that form nodules on their roots (or leaves).

The nodules of gymnosperms (orders Cycadales - cycads, Ginkgoales - hyikgs, Coniferales - conifers) have a branching coral-shaped, spherical or bead-like shape. They are thickened, modified lateral roots. The nature of the pathogen causing their formation has not yet been clarified. Endophytes of gymnosperms are classified as fungi (phycomycetes), actinomycetes, bacteria, and algae. Some researchers suggest the existence of multiple symbioses. For example, it is believed that in cycads, azotobacter, nodule bacteria and algae take part in symbiosis. The question of the function of nodules in gymnosperms has also not been resolved. A number of scientists are trying to primarily substantiate the role of nodules as nitrogen fixers. Some researchers consider podocarp nodules as water reservoirs, and the functions of aerial roots are often attributed to cycad nodules.

In a number of representatives of angiosperm dicotyledonous plants, nodules on the roots were discovered over 100 years ago.

First, let us dwell on the characteristics of the nodules of trees, shrubs and subshrubs (families Coriariacoae, Myricaceae, Betulaceae, Casuarinaceae, Elaeagnaceae, Rhamnaceae) included in this group. The nodules of most representatives of this group are coral-shaped clusters of pink-red color, becoming brown with age. There is evidence of the presence of hemoglobin in them. Species of the genus Elaeagnus (suckers) have white nodules.

Often the nodules have big sizes. In casuarinas they reach a length of 15 cm. They function for several years.

Plants with nodules are common in different climatic zones or are confined to a specific area. Thus, Shepherdia and Ceanothus are found only in North America, Casuarina - mainly in Australia. Suckers and sea buckthorn are much more widespread.

Many plants of this group grow on soils poor in nutrients - sands, dunes, rocks, swamps.

The nodules of alder (Alnus), in particular A. glutinosa, discovered back in the 70s of the last century by M. S. Voronin (Fig. 167), have been studied in more detail. There is an assumption that the nodules are characteristic not only of modern, but also extinct species of alder, since they were found on the roots of fossil alder in the tertiary deposits of the Aldana River valley - in Yakutia.

The endophyte in the nodules is polymorphic. It is usually found in the form of hyphae, vesicles and bacteroides (Fig. 168). The taxonomic position of the endophyte has not yet been established, since numerous attempts to isolate it into a pure culture have proven fruitless, and if cultures have been isolated, they have turned out to be non-virulent.

The main significance of this entire group of plants, apparently, lies in the ability to fix molecular nitrogen in symbiosis with an endophyte. Growing in areas where growing crops is economically unsustainable, they play the role of pioneers in the development of land. Thus, the annual increase in nitrogen in the soil of the dunes of Ireland (Cape Verde) under plantings of Casuarina equisetifolia reaches 140 kg/ha. The nitrogen content in the soil under alder is 30-50% higher than under birch, pine, and willow. Dried alder leaves contain twice as much nitrogen as the leaves of other woody plants. According to calculations by A. Virtanen (1962), an alder grove (on average 5 plants per 1 m2) gives an increase in nitrogen of 700 kg/ha over 7 years.

Nodules are found much less frequently in representatives of the Zygophyllaceae family. They were first discovered by B. L. Isachenko (1913) on the root system of Tribulus terrestris. Later, nodules were found in other Tribulus species.

Most members of the family Zygophyllaceae are xerophytic shrubs or perennial herbs. They are common in deserts of tropical and subtropical regions, and also grow on sand dunes, heaths and swamps of the temperate zone.

It is interesting to note that tropical plants such as vermilion parafolia form nodules only when high temperature and low soil moisture. Moistening the soil to 80% of its full moisture capacity prevents the formation of nodules. As is known, the opposite phenomenon is observed in leguminous plants of temperate climates. If there is insufficient moisture, they do not form nodules.

Nodules in plants of the parifolium family vary in size and location on the root system. Large nodules usually develop on the main root and close to the soil surface. Smaller ones are located on the lateral roots and at greater depths. Sometimes nodules form on stems if they lie on the surface of the soil.

The nodules of ground Tribulus on the sands along the Southern Bug look like small white, slightly pointed or round warts.

They are usually covered with a plexus of fungal hyphae penetrating into the root cortex.

In bright red parfolia, the nodules are the terminal thickenings of the lateral roots of plants. Bacteroides are found in the nodules; The bacteria are very similar to nodule bacteria.

The nodules of tropical plants Tribulus cistoides are hard, round, about 1 mm in diameter, connected to the roots by a wide base, and are often whorled on old roots. More often they are located on the roots, alternating on one or both sides (Fig. 169). Nodules are characterized by the absence of a meristem zone. A similar phenomenon is observed during the formation of nodules in coniferous plants. The nodule therefore arises due to the division of cells of the stele pericycle.

A histological study of Tribulus cistoides nodules at different stages of development showed that they lack microorganisms. Based on this, as well as the accumulation of large amounts of starch in the nodules, they are considered formations that perform the function of providing plants with reserve nutrients.

Nodules of forest reed grass are spherical or somewhat elongated formations up to 4 mm in diameter, sitting tightly on the roots of plants (Fig. 170). The color of young nodules is most often white, occasionally pinkish, while old ones are yellow and brown. The nodule is connected to the central cylinder of the root by a wide vascular bundle. Like Tribulus cistoides, reed grass nodules have a cortex, cortical parenchyma, endodermis, pericyclic parenchyma and a vascular bundle (Fig. 171).

The bacteria in the nodules of forest reed grass are very similar to the nodule bacteria of leguminous plants.

Nodules were found on the roots of cabbage and radish - representatives of the cruciferous family. It is assumed that they are formed by bacteria that have the ability to bind molecular nitrogen.

Among plants of the madder family, nodules are found in the coffee plants Coffea robusta and Coffea klainii. They branch dichotomously, are sometimes flattened and have the appearance of a fan. Bacteria and bacteroid cells are found in the tissues of the nodule. The bacteria, according to Steyaert (1932), belong to Rhizobium, but were named Bacillus coffeicola.

Nodules in plants of the rose family were found on dryad (partridge grass). Two other representatives of this family, Purshia tridentata and Cercocarpus betuloides, have typical coral-shaped nodules. However, there is no data in the literature on the structure of these nodules and the nature of their causative agent.

From the heather family, only one plant can be mentioned - bear ear(or bearberry), which has nodules on the root system. Many authors believe that these are coral-shaped ectotrophic mycorrhizae.

In angiosperm monocots, nodules are common among representatives of the grass family: meadow foxtail, meadow bluegrass, Siberian volosnets and saltmarsh volosnets. Nodules form at the ends of roots; They can be oblong, round, spindle-shaped. In foxtail, young nodules are light, transparent or translucent, and with age they acquire a brown or black color. Data on the presence of bacteria in nodule cells are contradictory.

Leaf nodules.

There are over 400 known species of different plants that form nodules on leaves. The most well studied are the nodules of Pavetta and Psychotria. They are located on the lower surface of the leaves along the main vein or scattered between the lateral veins, have an intense green color. Chloroplasts and tannin are concentrated in the nodules. With aging, cracks often appear on the nodules.

The formed nodule is filled with bacteria that infect the leaves of the plant, apparently at the time of seed germination. When sterile seeds are grown, nodules do not appear and the plants develop chlorotic. Bacteria isolated from leaf nodules of Psychotria bacteriopbyla turned out to belong to the genus Klebsiella (K. rubiacearum). Bacteria fix nitrogen not only in symbiosis, but also in pure culture - up to 25 mg of nitrogen per 1 g of sugar used. It must be assumed that they play an important role in the nitrogen nutrition of plants on infertile soils. There is reason to believe that they supply plants not only with nitrogen, but also with biologically active substances.

Sometimes glossy films or multi-colored spots can be seen on the surface of the leaves. They are formed by phyllosphere microorganisms - a special type of epiphytic microorganisms that also participate in nitrogen nutrition of plants. Phyllosphere bacteria are predominantly oligonitrophilic (they live on trace amounts of nitrogen-containing compounds in the environment and, as a rule, fix small amounts of molecular nitrogen) and are in close contact with the plant.

Biological encyclopedic dictionary

Genus nitrogen-fixing bacteria, forming nodules on the roots of many legume plants. They absorb atmospheric molecular nitrogen and convert it into nitrogen compounds that are absorbed by plants, which, in turn, will provide other plants... ... Ecological dictionary

A genus of bacteria that forms nodules on the roots of many leguminous plants and fixes molecular nitrogen in the air in symbiosis with the plant. They do not form spores, they are aerobes. Enrich the soil with nitrogen. See also Nitrogen fixation... Big Encyclopedic Dictionary

Cross section of a soybean root nodule. Bacteria, lat. Bradyrhizobium japonicum, inoculate roots and enter into a nitrogen-fixing symbiosis. Nodule bacteria... Wikipedia - symbiont bacteria that develop on the tissues of the roots of legumes and some other plants, capable of binding free nitrogen from the air and making it available to higher plants … Dictionary of botanical terms

Lives on the roots of legumes. and form special nodules on them, the size of poppy seeds to beans and larger. K.b. are important factor increasing productivity, because with their help, legumes grow. absorb free nitrogen from the atmosphere... Agricultural dictionary-reference book

NODULE BACTERIA- (Rhizobium), a genus of aerobic bacteria that settle in nodules on the roots of legumes and have the ability to absorb atmosphere. nitrogen and enrich the soil with it. They live in symbiosis with plants, providing them with nitrogen and receiving carbon products from plants... Agricultural Encyclopedic Dictionary

nodule bacteria- (Rhizobium), a genus of aerobic bacteria that settle in nodules on the roots of leguminous plants and have the ability to absorb atmospheric nitrogen and enrich the soil with it. They live in symbiosis with plants, providing them with nitrogen and receiving from plants... ... Agriculture. Large encyclopedic dictionary

Nodule bacteria were the first group of nitrogen-fixing microbes that humanity learned about.

About 2,000 years ago, farmers noticed that cultivating legumes restored fertility to depleted soil. This special property of legumes was empirically associated with the presence of peculiar nodules, or nodules, on their roots, but for a long time they could not explain the reasons for this phenomenon.

Much more research was needed to prove the role of legumes and bacteria living on their roots in fixing atmospheric nitrogen gas. But gradually, through the work of scientists from different countries, the nature was revealed and the properties of these wonderful creatures were studied in detail.

Nodule bacteria live with leguminous plants in symbiosis, that is, they bring each other mutual benefit: the bacteria absorb nitrogen from the atmosphere and convert it into compounds that can be used by plants, and they, in turn, supply the bacteria with substances containing carbon, which wounds absorb from the air in the form of carbon dioxide.

Outside the nodules on artificial nutrient media, nodule bacteria can develop at temperatures from 0 to 35°, and the most favorable (optimal) temperatures for them are about 20-31°. The best development of microorganisms is usually observed in a neutral environment (at a pH of 6.5-7.2).

In most cases, the acidic reaction of the soil negatively affects the vital activity of nodule bacteria; in such soils, inactive or ineffective (not fixing air nitrogen) races are formed.

The first researchers of root nodule bacteria assumed that these microbes could settle on the roots of most types of legumes. But then it was found that they have a certain specificity, have their own “tastes” and “rent” future “housing” in strict accordance with their needs. This or that race of nodule bacteria can enter into symbiosis with leguminous plants only of a certain species.

Currently, nodule bacteria are divided into the following groups (according to the host plants on which they settle):

• nodule bacteria of alfalfa and sweet clover;
• clover nodule bacteria;
• nodule bacteria of peas, vetch, china and broad beans;
• soybean nodule bacteria;
• nodule bacteria of lupine and seradella;
• bean nodule bacteria;
• nodule bacteria of peanuts, cowpeas, cowpeas, etc.

It must be said that the specificity of nodule bacteria in different groups is not the same. Picky “tenants” sometimes lose their scrupulousness. While clover nodule bacteria are distinguished by very strict specificity, the same cannot be said about pea nodule bacteria.

The ability to form nodules is not characteristic of all legumes, although in general it is widespread among representatives of this huge family. Of the 12 thousand species of legumes, 1063 were specially studied. It turned out that 133 of them are not capable of forming nodules.

The ability to symbiose with nitrogen fixers is apparently not unique to leguminous plants, although they are the only important nitrogen-fixing crops in agriculture. It has been established that atmospheric nitrogen is bound by bacteria living in nodules on the roots of oleaster, sea buckthorn, shepherdia, radiata pine, footcarp, hedgehog, and subtropical plants of the genus casuarina. Bacteria living in the leaf nodes of some tropical shrubs are also capable of nitrogen fixation.

Nitrogen fixation is also carried out by actinomycetes living in the nodules of alder roots, and, possibly, by fungi living in the roots of ryegrass and some heather plants.

But for agriculture, legumes are, of course, of greatest practical interest. Most of the noted non-legume plants are of no agricultural importance.

A very important question for practice is: how do nodule bacteria live in the soil before they infect the roots?

It turns out that nodule bacteria can survive in the soil for a very long time in the absence of “hosts” - legumes. Let's give an example. At the Moscow Agricultural Academy named after K. A. Timiryazev there are fields laid out by D. N. Pryanishnikov. The same crops are grown on them year after year and permanent fallow is maintained, on which no plants have been grown for almost 50 years. Analysis of the soils of this fallow and the field of permanent rye showed that nodule bacteria were found in them in significant quantities. Under permanent rye there are several more of them than in a couple.

Consequently, nodule bacteria survive the absence of leguminous plants relatively well and can wait a very long time to meet them. But under these conditions they lose their remarkable ability to fix the bunker. However, bacteria with “pleasure” stop their “free lifestyle”, as soon as a suitable legume plant comes across their path, they immediately penetrate the roots and create their own nodule houses.

Three factors take part in the complex process of nodule formation: two living organisms - bacteria and plants, between which close symbiotic relationships are established, and conditions external environment. Each of these factors is an active participant in the process of nodule formation.

One of important features nodule bacteria is their ability to secrete so-called stimulating substances; these substances cause rapid growth of root tissue.

Another significant feature is their ability to penetrate the roots of certain plants and cause the formation of nodules, in other words, their infectious ability, which, as already mentioned, varies among different races of nodule bacteria.

The role of a legume plant in the formation of nodules is determined by the ability of plants to secrete substances that stimulate or inhibit the development of bacteria.

The susceptibility of a legume plant to infection by nodule bacteria is greatly influenced by the content of carbohydrates and nitrogenous substances in its tissues. The abundance of carbohydrates in the tissues of a legume plant stimulates the formation of nodules, and an increase in nitrogen content, on the contrary, inhibits this process. Thus, the higher the C/N ratio in the plant, the better the development of nodules.

It is interesting that the nitrogen contained in plant tissues seems to interfere with the introduction of “intruder” nitrogen.

The third factor - external conditions (lighting, batteries, etc.) also has a significant impact on the process of nodule formation.

But let us return to the characteristics of individual types of nodule bacteria.

Infectious ability, or the ability to form nodules, does not always indicate how actively nodule bacteria fix atmospheric nitrogen. The “performance” of nodule bacteria in fixing nitrogen is often called their efficiency. The higher the efficiency, the greater the efficiency of these bacteria, the more valuable they are for the plant, and therefore for agriculture in general.

Races of nodule bacteria, effective, ineffective, and transitional between these two groups, are found in the soil. Infection of leguminous plants with an effective race of nodule bacteria promotes active nitrogen fixation. An ineffective race causes the formation of nodules, but nitrogen fixation does not occur in them, therefore, building material is wasted in vain, the plant feeds its “guests” for nothing.

Are there differences between effective and ineffective races of nodule bacteria? So far, no such differences in form or behavior on artificial nutrient media have been found. But the nodules formed by effective and ineffective races show some differences. There is, for example, an opinion that efficiency is related to the volume of root tissue infected with bacteria (in effective races it is 4-6 times greater than in ineffective ones) and the duration of the functioning of these tissues. In tissues infected with effective bacteria, bacteroids and a red pigment are always found, which is completely identical to blood hemoglobin. It is called leghemoglobn. Ineffective nodules have a smaller volume of infected tissue, they lack leghemoglobin, bacteroids are not always detected and they look different than in effective nodules.

These morphological and biochemical differences are used to isolate effective races of nodule bacteria. Typically, bacteria isolated from large, well-developed nodules that are pinkish in color are very effective.

It was already said above that the “work” of nodule bacteria and its “efficiency” depends on a number of external conditions: temperature, environmental acidity (pH), lighting, oxygen supply, soil content nutrients etc.

The influence of external conditions on the fixation of atmospheric nitrogen by nodule bacteria can be demonstrated using several examples. Thus, the content of nitrate and ammonia salts in the soil plays a significant role in the efficiency of nitrogen fixation. In the initial phases of legume plant development and nodule formation, the presence of small amounts of these salts in the soil has a beneficial effect on the symbiotic community; and later the same amount of nitrogen (especially its nitrate form) inhibits nitrogen fixation.

Consequently, the richer the soil in nitrogen available to the plant, the weaker the nitrogen fixation. The nitrogen contained in the soil, as well as in the body of the plant, seems to prevent the attraction of new portions from the atmosphere. Among other nutrients, molybdenum has a noticeable effect on nitrogen fixation. When this element is added to the soil, more nitrogen accumulates. This is apparently explained by the fact that molybdenum is part of the enzymes that fix atmospheric nitrogen.

It has now been reliably established that legumes grown in soils containing insufficient amounts of molybdenum develop satisfactorily and form nodules, but do not absorb atmospheric nitrogen at all. Optimal quantity molybdenum for effective nitrogen fixation is about 100 g of sodium molybdate per 1 ha.

The role of legumes in increasing soil fertility

So, legumes are very important for increasing soil fertility. By accumulating nitrogen in the soil, they prevent the depletion of its reserves. The role of legumes is especially important in cases where they are used for green fertilizers.

But agricultural practitioners are naturally also interested in the quantitative side. How much nitrogen can be accumulated in the soil when cultivating certain legumes? How much nitrogen remains in the soil if the crop is completely removed from the field or if the legumes are plowed under as green manure?

It is known that if legumes are infected with effective races of nodule bacteria, they can bind from 50 to 200 kg of nitrogen per hectare of crop (depending on the soil, climate, plant type, etc.).

According to the famous French scientists Pochon and De Berjac, under normal field conditions, legumes fix approximately the following amounts of nitrogen (in kg/ha):

Root remains of annual and perennial leguminous plants in different conditions crops and on different soils contain various quantities nitrogen. On average, alfalfa leaves about 100 kg of nitrogen per hectare in the soil annually. Clover and lupine can accumulate approximately 80 kg of bound nitrogen in the soil; annual legumes leave up to 10-20 kg of nitrogen per hectare in the soil. Taking into account the areas occupied by legumes in the USSR, the Soviet microbiologist E. N. Mishustin calculated that they return about 3.5 million tons of nitrogen to the fields of our country annually. For comparison, we point out that our entire industry produced 0.8 million tons in 1961 nitrogen fertilizers, and in 1965 it will give 2.1 million tons. Thus, nitrogen extracted from the air by legumes in symbiosis with bacteria occupies a leading place in the nitrogen balance of agriculture in our country.

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 proof of their high degree of adaptability to various soil and climatic conditions and the ability to lead a symbiotic and saprophytic way of life.

By schematizing the currently available data on the distribution of nodule bacteria in nature, the following generalizations can be made.

In virgin and cultivated soils, nodule bacteria are usually present in large quantities of those types of leguminous plants that are part of the wild flora or have been cultivated for a long time in the area. The number of nodule bacteria is always highest in the rhizosphere of leguminous plants, somewhat less in the rhizosphere of other species and low 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)

In nature and agricultural practice, cross-infection of different types of leguminous plants often leads to the appearance of nodules on the roots that are not sufficiently active in fixing molecular nitrogen. This usually depends on the absence of the corresponding species 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 bacteria of the Cross groups, or develop without nodules.

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

Nodule bacteria are used for the industrial production of nitragine, which is used to treat legume seeds. They were first discovered by M. S. Voronin in 1866. Later, by M. V. Beyerinck (1888), they were isolated 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 internal integument of the root, into the parenchyma, causing increased cell division and proliferation. Ugly growths called nodules or nodules form on the roots. Initially, bacteria absorb the plant’s nutrients and somewhat inhibit its growth. Then, as the nodule tissue grows, a symbiosis is established between the bacteria and higher plants. Bacteria receive carbonaceous food (sugars) and minerals from the plant, and in return provide it with nitrogenous compounds.

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

Nodule bacteria supply the legume plant with nitrogen. The plant uses this fixed nitrogen and, in turn, supplies the nodule bacteria with the carbon-containing organic substances they need. Nodule bacteria can use various sugars and alcohols as a carbon source.

Nodule bacteria are microaerophiles (they develop in small amounts of oxygen in the environment), however, they prefer aerobic conditions. Nodule bacteria emerging 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, liming of the soil is necessary. Inoculation without liming has very little effect on yield and protein content.

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

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 microns, mobile, multiply by division

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



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

In modern species of leguminous plants, nodules are found on the roots of many representatives 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 detected in only approximately 1,300 species (243 genera). This primarily includes plant species used in agriculture (more than 200).

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

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

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

20 years later, the Dutch scientist Beijerin isolated bacteria from the nodules of peas, vetch, china, beans, seradella and commonweed and studied their properties, testing their ability to infect plants and cause the formation of nodules. He named these microorganisms Bacillus radicicola. Since the genus Bacillus includes bacteria that form spores, 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 literature today.

To designate a species of nodule bacteria, it is customary to add to the generic name Rhizobium a term 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 - nodule bacteria of clover, Rhizobium lupini - nodule bacteria of lupine, etc. In cases where nodule bacteria are capable of forming nodules on the roots of different types of leguminous plants, i.e., causing so-called cross-infection, the species name is as if collective - it reflects precisely this “cross-infecting” ability. For example, Rhizobium leguminosarum - nodule bacteria of peas (Pisum), lentils (Lens), and chin (Lathyrus).

Morphology and physiology of nodule bacteria. Nodule bacteria are characterized by an amazing variety of forms - polymorphism. Many researchers paid attention to this when studying nodule bacteria in pure culture in laboratory conditions and in soil. Nodule bacteria can be rod-shaped or oval. Among these bacteria there are also filterable forms, L-forms, coccoid immobile and motile 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, and reproduce by division. In the rod-shaped cells of clover nodule bacteria, division by lacing is observed. With age, rod-shaped cells may progress 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 turn into the state of so-called girdled rods. They received this name due to the alternation of dense and loose sections of protoplasm in the cells. The banding of cells is clearly visible when viewed under a light microscope after treatment of cells with aniline dyes. Dense areas of protoplasm (belts) 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 (Fig. 143, 1). The belts can be located in the middle of the cage or at the ends. The girdling of cells is also visible on electron diffraction patterns if the preparation is not treated with contrasting substances before viewing (Fig. 143, 3). Probably, with age, the bacterial cell becomes filled with fatty inclusions that do not perceive color and, as a result, cause the cell to become striated. The stage of “girdled rods” precedes the stage of formation of bacteroids - cells of irregular shape: thickened, branched, spherical, pear-shaped and flask-shaped (Fig. 144). The term “bacteroids” was introduced into the literature by J. Brunkhorst in 1885, applying it to formations of unusual shape, much larger than rod-shaped bacterial cells, found in the tissues of nodules.

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

In old (two-month) 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 different types of legumes grow at different rates. The fast-growing ones include nodule bacteria of peas, clover, alfalfa, broad beans, vetch, lentils, china, sweet clover, fenugreek, beans, chickpeas, sweet grass; slow-growing - nodule bacteria of lupine, soybean, peanut, seradella, mung bean, cowpea, sainfoin, gorse. Fully formed colonies of fast-growing crops can be obtained on the 3rd - 4th day of incubation, colonies of slow-growing ones - on the 7th - 8th.

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

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

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Colonies of fast-growing nodule bacteria are the color of baked milk, often translucent, slimy, with smooth edges, moderately convex, and grow over time 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 in small amounts of oxygen in the environment), however, they prefer aerobic conditions.

Nodule bacteria use carbohydrates and organic acids as a source of carbon in nutrient media, and various mineral and organic nitrogen-containing compounds as a source of nitrogen. When cultivated in media with a high content of nitrogen-containing substances, nodule bacteria may lose the ability to penetrate the plant and form nodules. Therefore, nodule bacteria are usually grown on plant extracts (bean, pea broth) or soil extracts. Nodule bacteria can obtain the phosphorus necessary for development 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 optimal temperature is required in the range of 24-26°. At 0° and 37°C, growth stops. Typically, cultures of nodule bacteria are stored in laboratory conditions at low temperatures (2-4 °C).

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

All nodule bacteria are approximately equally resistant to an alkaline reaction of the environment (pH = 8.0), but are not equally sensitive to an acidic environment.

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 in itself specific - 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 capable of infecting only a certain, sometimes large, sometimes smaller, group of leguminous plants, and in this sense, the specificity of nodule bacteria is their 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 non-alkaloid varieties of lupine). With broad specificity, nodule bacteria of peas can infect pea, china, and bean plants, and nodule bacteria of china and beans can infect pea plants, i.e., they are all characterized by the ability of “cross-infection.” 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 a different adaptability of nodule bacteria to plants within the cross-infection group. Thus, nodule bacteria in alfalfa can form nodules in sweet clover. But nevertheless, they are more adapted to alfalfa, and sweet clover bacteria - to sweet clover.

In the process of infection of the root system of leguminous plants by nodule bacteria, it is of great importance 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 root tissue, multiply there and cause the formation of nodules.

An important role is played not only by the ability to penetrate into the roots of a 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 ensures more vigorous infection of the roots compared to other strains, resulting 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, it should be taken into account competitive ability, which often masks the property of virulence in natural conditions.

It is necessary that virulent strains also have competitiveness, that is, 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 it forms 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, in symbiosis with leguminous plants, to assimilate molecular nitrogen and satisfy the needs of the host plant. Depending on the extent to which nodule bacteria contribute to increasing the yield of legumes (Fig. 146), they are usually divided into active (effective), inactive (ineffective) and inactive (ineffective).

A strain of bacteria that is inactive for one host plant in symbiosis with another type of legume 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 constant property. Often in laboratory practice, a loss of activity is observed 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 and amino acids. One of the reasons for the loss of activity of nodule bacteria may be the influence of phage. By passaging, i.e., repeatedly passing bacteria through a host plant (adaptation to a specific 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 changing 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 by nodule bacteria, it is necessary to have a fairly large number of viable bacterial cells in the root zone. Researchers have different opinions regarding the number of cells required to ensure the inoculation process. Thus, according to the American scientist O. Allen (1966), for inoculation of small-seeded plants, 500-1000 cells are required, for inoculation of large-seeded plants - at least 70,000 cells per 1 seed. According to the Australian researcher J. Vincent (1966), at the time of inoculation, each seed should contain at least several hundred viable and active cells of nodule bacteria. There is evidence that single cells can invade root tissue.

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

In the rhizosphere of a legume 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 forms regardless of the presence of bacteria in the rhizosphere. This layer is clearly visible when examined under a light optical microscope (Fig. 147). After inoculation, nodule bacteria 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.

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

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

The hypothesis about the penetration of nodule bacteria into root tissue through root hairs is interesting and not without foundation. The route of passage of nodule bacteria through root hairs is recognized by most researchers.

The assumption 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 at 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 that the cells of nodule bacteria become smaller in the rhizosphere of legume plants confirm this position.

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

Recently, the auxin hypothesis has gained 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 root secretions of plants. The presence of heteroauxin is associated with the curvature of root hairs, which is usually observed when the root system is infected by nodule bacteria ( Fig. 150).

The source of β-indoleacetic acid at the time of plant infection is obviously not only plants that secrete tryptophan through the root system, which many types of bacteria, including nodule bacteria, can convert into β-indoleacetic acid. The 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 different plant species, not just legumes. At the same time, nodule bacteria cause curvature of root hairs only in leguminous plants, showing quite significant selectivity. If the effect in question were determined only by β-indoleacetic 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.

It should also be noted that in some cases, non-curved root hairs become infected. Observations show that in alfalfa and peas, 60-70% of root hairs bend and curl, and in clover - about 50%. In some types of clover, this reaction is observed in no more than 1/4 of the infected hairs. In the curvature reaction, the condition of the root hair is obviously of great importance. Growing root hairs are most sensitive to the action of substances produced by bacteria.

It is known that nodule bacteria 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 plants to synthesize the enzyme polygalacturonase. This enzyme, by destroying pectin substances, affects the membrane of root hairs, making it more plastic and permeable. Polygalacturonase is always present in small quantities 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 the root thanks to companion bacteria that produce pectinolytic enzymes. This hypothesis was put forward based on the following facts. When microscopying root hairs, many researchers noted the presence of a light spot around which nodule bacteria accumulate. This spot may be a sign of the beginning of maceration (destruction) of tissue by protopectinase, similar to 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 the root.

Another hypothesis should be noted, according to which nodule bacteria enter the root during the formation of a finger-like invagination of the surface of the root hair. The electron diffraction pattern of a section of a root hair, which confirms this hypothesis (Fig. 150, 3), shows a root hair curved in the shape of an umbrella handle, in the bend of which there is an accumulation of nodule bacteria. Nodule bacteria seem to be drawn in (swallowed) by the root hair (similar to pinocytosis).

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

The process of introduction of nodule bacteria into root tissue is the same for 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 occurs intensively. The duration of the phases varies 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 boundaries between phases. The most intensive penetration of nodule bacteria into root hairs occurs in the early stages of plant development. The second phase ends during the period of mass formation of nodules. Often, the introduction 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. At later stages of infection, the nodules are usually located lower along the root.

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

Having penetrated the root (through the root hair, epidermal cell, and sites of root damage), nodule bacteria then move into the plant root tissue. Bacteria most easily pass through intercellular spaces.

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

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

The term “infection thread” arose from studying the infection process under a light microscope. Beginning with the work of Beijerinck, the infection thread began to be viewed as a mucous hyphae-like mass with replicating bacteria enclosed within it.

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 bacteria penetrate the root.

The number of infected root hairs varies significantly among individual plants. Typically, infection threads appear in deformed, twisted root hairs. However, there are indications that similar threads are sometimes found in straight hairs. Most 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 several have common threads of infection, giving rise to one nodule (Fig. 151).

The percentage of infected root hairs in the total number of deformed ones 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 infection threads there are many (up to 80%) so-called abortive threads that have stopped developing. The rate of advancement of normally developing infection threads in a plant is 5 - 8 microns per hour. At this speed, the infection thread can travel through a root hair 100-200 microns long in one day.

Morphological and anatomical characteristics of nodules in their ontogenesis.

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

1st type - nodules arise during the division of cells of the pericycle (root layer), usually located opposite the protoxylem (the 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 a pathogen into the parenchyma cells of the cortex and endoderm (inner layer of the primary cortex) - 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 the zkzo- and endotypes, their development process 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 certain similarities in their formation. 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 of the development of lateral roots and nodules emphasize profound differences in the type of their formation. Lateral roots arise in the pericycle. From the very first moments of development, they are associated with the central cylinder of the main root, from which the central cylinders of the lateral roots branch, and they always arise against the ray of the primary wood. The formation of a nodule, unlike a 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 to the vessels of the root through the tracheid zone and have their own endoderm (Fig. 152).

The difference in the nature of the emergence of nodules 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 the nodules become visible very quickly after infection of the root by 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 core cells located at a distance of 2-3 layers from the ends of the infection threads. The layers of the cortex, penetrated by infectious threads, remain unchanged. At the same time, in Seradella, the division of bark cells occurs directly under the infected root hair, and in pea, cell division is observed only in the penultimate layer of the bark.

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

The divided cells of the cortex change: the nuclei become rounded and increase in size, the nucleoli especially increase. After mitosis, the nuclei disperse and, without taking their original shape, begin to divide again.

Bean salads

From the book Salads. Just. Fast. Tasty author Gorbacheva Ekaterina Gennadievna

Nodule weevils

From the book Legumes. We plant, grow, harvest, heal author Zvonarev Nikolai Mikhailovich

From the book Garden without pests author

Nodule pea weevils There are several types of them. The most common weevils in the CIS are striped and bristly (gray) weevils. They are easy to distinguish. The tabby has noticeable light stripes on its back, passing even through the elytra, the color is gray, the length

Nodule pea weevils

From the book Peas, Beans and Legumes author Fatyanov Vladislav Ivanovich

Nodule pea weevils There are several types of these pests. The most common in the CIS are striped and bristly (gray). They are easy to distinguish. The tabby has noticeable light stripes on its back running through the elytra, the general background is gray, the length

4.2. Kingdom of Bacteria. Features of structure and vital activity, role in nature. Bacteria are pathogens that cause diseases in plants, animals, and humans. Prevention of diseases caused by bacteria. Viruses

From the book Biology [Complete reference book for preparing for the Unified State Exam] author Lerner Georgy Isaakovich

4.2. Kingdom of Bacteria. Features of structure and vital activity, role in nature. Bacteria are pathogens that cause diseases in plants, animals, and humans. Prevention of diseases caused by bacteria. Viruses Basic terms and concepts tested in the exam paper:

Nodule bacteria

TSB

Nodule weevils

From the book Big Soviet Encyclopedia(KL) by the author TSB