Formation of symbiotic nodules on pea roots. Mycorrhiza

Nodules in non-legume plants. Root nodules or formations resembling nodules are widespread on the roots of not only leguminous plants. They are found in gymnosperms and angiosperms.[ ...]

It has been shown that the number of underground nitrogen-fixing root nodules in legumes (see Figure 4.3) is regulated by the photoperiod acting through the plant leaves. Nitrogen-fixing nodule bacteria need food energy for their functioning, and food is produced by plant leaves. The more light plants get and the more chlorophyll they contain, the more food bacteria can get. Thus, the photoperiod promotes maximum coordination between the activity of the plant and its partners - microorganisms.[ ...]

They are the main absorptive organs of the plant. Abundantly branched short roots often contain mycorrhiza. Podocarps have root nodules with bacteria resembling legume nodules, and such roots, with the exception of some genera, are equipped with root hairs. Root hairs in conifers are confined to such a narrow zone of the apex and fall off so easily when the root is washed that they are very often not noticed. The roots develop a multi-layered pericycle and a clearly expressed single-layered endoderm.[ ...]

The presence of leguminous plants in the grass mixture provides only a small part of the high nitrogen demand of cereals (because nitrogen is released from root nodules only very late and at too great a depth in the soil). It is believed that it is necessary to apply from 100 to 200 kg/ha of nitrogen in 3-4 doses, and the dose of 100 kg/ha is undoubtedly the minimum.[ ...]

The practically inexhaustible reservoir of atmospheric molecular nitrogen is inaccessible to the vast majority of living creatures. Biological nitrogen fixation is carried out by a very specialized group of anaerobic bacteria that inhabit the root nodules of leguminous plants. With the help of a special enzyme, these soil microbes carry out a reaction that, in industrial nitrogen fixation, requires an expensive catalyst, a temperature of 500 ° and a pressure of up to 1000 atmospheres. A certain amount of molecular nitrogen is oxidized to N0 during lightning discharges and photochemical reactions in the atmosphere.[ ...]

There are only 3 genera in the sucker family and about 55 species distributed in Europe, Asia and North America. Lokhovye - trees and shrubs with characteristic pubescence of corymbose scales or stellate hairs. Their leaves are alternate or sometimes opposite, like those of Shepherdia (Sierrieria), on short petioles, whole and whole-crowned, evergreen or falling. All three genera are characterized by the presence of root nodules with nitrogen-fixing bacteria, due to which suckers can also grow on very poor soils.[ ...]

So, only prokaryotes, non-nuclear, the most primitive microorganisms, can convert biologically useless gaseous nitrogen into the forms necessary for the construction and maintenance of living protoplasm. When these microorganisms form mutually beneficial associations with higher plants, nitrogen fixation is greatly enhanced. The plant provides the bacteria with a suitable habitat (i.e. root nodules), protects the microbes from excess oxygen that interferes with fixation, and supplies them with the high quality energy they need. For this, the plant receives easily digestible fixed nitrogen.[ ...]

Nitrogen fertilizers have become very expensive due to the decline in fossil fuel production, and there has been increasing public and political concern about the possibility of chemical pollution. Consequently, attention is now focusing on nitrogen fixation as an alternative to nitrogen fertilization. Importance of nitrogen fixation for Agriculture led to intensive research on bacteria capable of entering into a symbiotic relationship with legumes. One of these bacteria are bacteria of the genus Rhizobium, which were isolated from the root nodules of various legumes, such as peas, lupins, clover, soybeans, and alfalfa.

Nitrogen is an essential factor in soil fertility. It is part of the vital compounds - amino acids, proteins, nucleic acids.

The Earth's atmosphere contains a colossal amount of nitrogen - 79.2 percent, but it is not available to plants. For plants, it is not atmospheric nitrogen that is important, but the nitrogen contained in the soil. Meanwhile, the reserves of nitrogen forms available to plants, even in chernozem soils, are not so large as to ensure uninterrupted high yields of agricultural crops.

In the soil, nitrogen is in a bound state mainly in the form of salts of nitric acid (nitrates) and ammonium salts. Back in the 17th century, the German chemist Johann Glauber (1604-1670) drew attention to the exceptional importance of these compounds for plant growth. In his writings, he called saltpeter the salt of fertility. Intensive exploitation of deposits of Chilean saltpeter began. Mankind has mastered the artificial fixation of atmospheric nitrogen and created a powerful nitrogen industry.

However, the huge scale industrial production bound nitrogen give us not only confidence in sustainable high yields, but also justified concern in connection with pollution natural environment nitrogen compounds. Excessive application of nitrogen fertilizers to the soil disrupts the natural cycle of substances. In addition, plants assimilate only 40-50 percent of the nitrogen introduced into the soil, and the rest ends up in water bodies and ground water causing them to become contaminated. Nitrates and nitrites are extremely hazardous to human health.

In connection with the severity of the nitrate problem, scientists are even more persistently studying the processes of natural fixation of atmospheric nitrogen by nodule bacteria, as well as some free-living microorganisms.

It is thanks to these microorganisms that the nitrogen content in soils in the case of their rational use persists or even slightly increases. On each hectare of soil occupied by leguminous plants with nodules on their roots, from 100 to 250 kilograms of atmospheric nitrogen are recorded. Part of it is used by the legumes themselves for the synthesis of nitrogen-containing substances (amino acids, proteins, nucleic acids.), And about 30 percent remains with crop residues in the soil, thereby increasing its fertility.

Of all living organisms, only a few genera of bacteria are capable of fixing atmospheric nitrogen. The best known of these is the symbiotic bacterium Rhizobium, which forms nodules on the roots of legumes and some other plants.

In 1866, the famous botanist and soil scientist M. S. Voronin saw the smallest "calves" in the nodules on the roots of leguminous plants. Voronin put forward bold assumptions for that time: he linked the formation of nodules with the activity of bacteria, and the increased division of root tissue cells with the reaction of the plant to bacteria that penetrated the root. Twenty years later, the Dutch scientist Beijerinck isolated bacteria from the nodules of peas, vetch, chiny, beans, seradella, and lollipops and studied their properties, testing their ability to infect plants and cause the formation of nodules.

Nodule bacteria can be rod-shaped and oval. Of the 13,000 species (550 genera) of leguminous plants, the presence of nodules has so far been identified only in approximately 1,300 species (243 genera). This primarily includes plant species used in agriculture (more than 200).

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

Thus, leguminous plants and nodule bacteria are in a state of symbiosis.

Nodule bacteria are microaerophiles (they develop with small amounts of oxygen in the environment), however, they prefer aerobic conditions (aerobes). If we talk about nodule bacteria in general, then for them the formation of nodules only in a group of leguminous plants is already specific in itself - they have selectivity for leguminous plants. Among them there are those that are able to infect only a certain, sometimes large, sometimes smaller, group of leguminous plants.

The specificity of nodule bacteria can be narrow (clover nodule bacteria infect only a group of clovers). With a wide specificity, pea nodule bacteria can infect pea, chin, and bean plants, and pea and bean nodule bacteria can infect pea plants, i.e., they are all characterized by the ability to “cross-infect”.

In connection with the foregoing, it seemed interesting to us to trace the process of formation of symbiotic nodules on the roots of the pea Pisum sativum.

Goals and objectives of the study:

1. Study the literature on the research topic.

2. Develop a methodology for conducting the experiment.

3. Find out at what stage of development of the Pisum sativum pea plant visible symbiotic nodules appear on its roots.

4. Make visual aids for biology lessons.

The novelty of the work is that we invented and tested a method for observing the formation of nodules on pea roots. The applied value of the work is that prepared preparations of pea roots with nodules are used as demonstrative material in botany lessons.

Main part

2. 1. Experimental technique

The pea plant Pisum sativum of the varieties "Oregon" and "Alpine Emerald" was chosen as the object of study.

The life form of peas is an annual grass, which makes it possible to obtain the results of the experiment in a relatively short period of time (2 months).

To exclude the influence of adverse conditions, mainly weather, during the growing season of the plant, it is advisable to sow the seeds in closed ground (in a greenhouse, on a windowsill or loggia). You can use one large or several small containers filled with the same earth mixture.

As a substrate for growing plants, you can take ordinary soil from a garden plot, or ready-made soil from a garden center.

Soak seeds in water before sowing. Plant the hatched seeds in the ground, water. Every 2 days after the emergence of seedlings, remove one plant at a time, free the roots from soil particles, washing with water.

It is necessary to carefully examine the roots of plants and record all the changes that occur in the process of growing peas.

2. 2. The course of the experiment. Results and discussions

Stage number 1. Preparation of seeds for sowing.

On July 1, I took Oregon pea seeds and put them under cheesecloth. I filled them with water so that the water almost completely covered the seeds, but at the same time they could breathe. They were green.

The shops in the city sell not only the Oregon variety. I also bought Alpine Emerald, which, for comparison, I put in another saucer. Although this variety is called "Alpine Emerald", the color of its seeds is yellow. It probably got its name because of the emerald color of the seeds, which are in a state of milky ripeness. The seeds are larger than those of the Oregon variety.

One bag contains about 15 seeds. I took four bags in total, two bags of each type. So many seeds were needed just in case not all the seeds would germinate.

Dry seeds are hard and shriveled. By evening, the pea seeds swelled, became soft, that is, from a dormant state, they passed into the swelling stage.

Stage number 2. Observation of seed germination.

On July 6, 5 days after the start of the experiment, the seeds became larger, the seed coat became smooth. I changed the water more often to release substances that inhibit germination. The sprouts are not visible yet. The color of the seeds is dominated by a green pigment. It was hot outside, t=27oC and the air temperature on the windowsill was high, which contributed to the germination of seeds. After all, as you know, for the germination of seeds, water, air and optimum temperature.

On July 7, Oregon seeds doubled in size. Seeds of the variety "Alpine Emerald" gradually become brighter and larger. Their size is one and a half times larger than the day before.

According to the literature, the time from sowing to the appearance of sprouts can be up to three weeks.

Light swellings appeared on the surface of the seeds - the beginning of the pecking phase, but they did not appear in all seeds.

In the Oregon variety, they formed in half of the seeds (50% germination).

In the Alpine Emerald variety, light swellings are larger in size, but in a smaller number of seeds (30% germination).

Seeds absorb water well. If the water was changed at night, the next one had to be added again in the morning, despite the cool temperature at night of about 18 oC.

The pecking phase occurred earlier than described in the literature I read - a week after soaking the seeds.

On July 8, roots appeared at the seeds, they are white, light. In the Alpine Emerald variety, they are larger than in the Oregon variety. This is due to the fact that the seeds of the "Alpine Emerald" are larger, therefore, contain more reserve nutrients.

Stage number 3. Planting pea seeds in the soil.

10 July. Today I planted pea seeds in Terra Vita soil. The pecking phase began in both varieties on the 10th day.

In total, I planted 17 seeds, of which 5 varieties are "Alpine Emerald" and 12 varieties are "Oregon".

First, I poured soil into each pot (it took 2 bags of earth), made a hole in each pot and put a seed in each hole with a root down.

I covered the seed with soil and sprinkled it with water to moisten it.

The length of the seedling roots was approximately 4 mm. You need to plant them carefully so that they do not break.

After 5 days, seedlings emerged from the soil.

The maximum height of plants is 2.5 cm.

The minimum height of plants is 0.5 cm.

Both varieties develop approximately the same, but still noticeably ahead of development in the Alpine Emerald variety.

2 weeks after sowing, the height of the plants reaches 10 cm. The first true leaves with tendrils appeared.

The tap root system of the plant has many lateral roots. At this stage, there are no visible changes in the root system associated with the penetration of bacteria.

According to the literature data, bacteria of the genus Risobium begin to penetrate into the roots of legumes already at the seedling stage.

There are a number of hypotheses about the mechanism of penetration of nodule bacteria into the plant root.

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

It is possible that nodule bacteria can penetrate into the root through the epidermal cells of young root tips. According to

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

Nodule bacteria are known to cause softening of the walls of root hairs. However, they do not form either cellulase or pectinolytic enzymes. In this regard, it was suggested that nodule bacteria penetrate the root due to the secretion of mucus of a polysaccharide nature, which causes the synthesis of the polygalacturonase enzyme by plants. This enzyme, destroying pectin substances, affects the shell of root hairs, making it more plastic and permeable.

The process of introduction of nodule bacteria into the root tissue is the same in all types of leguminous plants and consists of two phases. In the first phase, infection of the root hairs occurs. In the second phase, the process of nodule formation proceeds intensively. The duration of the phases is different for different types plants: in Trifolium fragiferum the first phase lasts 6 days, in Trifolium nigrescens - 3 days. In some cases it is very difficult to detect the boundaries between phases. The most intensive introduction of nodule bacteria into root hairs occurs at the early stages of plant development. The second phase ends during the mass formation of nodules.

In order not to miss the beginning of the second phase - the formation of visible nodules, we had to extract one plant from the soil every two days, carefully examine the roots and record the changes. In photo No. 2 - the appearance of a seedling and photo No. 3 - the formation of the first true leaves. No nodules were found during this period. In photo No. 4, the length of the stem is 15 cm, the length of the roots is 15 cm. No visible changes were found on the roots. In photo No. 5, the length of the root has not changed, but new lateral roots have appeared. The length of the stem is more than 20 cm, the first flower has appeared.

In photo No. 6, the root length is 25 cm. On the lateral roots, after washing them with water, swellings - nodules are clearly visible.

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

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

Essentially, an infection thread is a colony of multiplied bacteria. In annual plants, infection threads usually appear during the first period of infection of the root, in perennial plants - during long period development.

Bacteria can be released from the infection thread at different times and in different ways.

The vascular system of the nodule provides a link between the bacteria and the host plant. transported through the vascular bundles nutrients and exchange products.

Nodule bacteria that have emerged from the infection thread continue to multiply in the host tissue. The bulk of bacteria multiply in the cytoplasm of the cell, and not in the infection thread. Infected cells give rise to future bacteroid tissue.

Filled with rapidly multiplying cells of nodule bacteria, plant cells begin to intensively divide. After plant cells are completely filled with bacteria, mitosis stops. However, the cells continue to increase in size and are often highly elongated, leading to the formation of tumor-like nodules. The bacteroid zone of a nodule occupies its central part and makes up from 16 to 50% of the total dry mass of nodules.

When growing peas, it was necessary to create conditions without which the formation of nodules is impossible. One of the most important is adequate watering.

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

Since the soil substrate "Terra Vita" does not have sufficient moisture capacity, it was necessary to water frequently as the soil dried out.

Excess moisture, as well as its lack, is also unfavorable for symbiosis - due to a decrease in the degree of aeration in the root zone, the supply of oxygen to the root system of the plant worsens. Insufficient aeration also negatively affects nodule bacteria living in the soil, which, as you know, multiply better when oxygen is available.

With the help of litmus paper, we checked the soil in which peas were grown, since in acidic soils, as noted by A. V. Peterburgsky, aluminum and manganese salts pass into the soil solution, which have an unfavorable effect on the development of the root system of plants and the process of nitrogen absorption, as well as the content of digestible forms of phosphorus, calcium, molybdenum and carbon dioxide decreases.

The temperature factor plays an important role in the relationship between nodule bacteria and leguminous plants. The maximum nitrogen fixation of a number of leguminous plants is observed at 20-25°C. Temperatures above 30°C adversely affect nodule bacteria.

This condition was met by growing a pea plant on a windowsill, where there are no sharp fluctuations in air and soil temperatures.

Therefore, the formation of nodules is the result of complex phenomena that begin outside the root. Following the initial phases of infection, the formation of a nodule is induced, then the spread of bacteria in the nodule tissue zone and nitrogen fixation occur.

The timing of the appearance of the first visible nodules on the roots of various 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. The nodules of sickle alfalfa appear after 10 days.

Pisum Sativum pea nodules appeared on the 22nd day after sowing the seeds. We did not find significant differences in the timing of the appearance of nodules in the Alpine Emerald and Oregon varieties.

Nodules on the roots of peas are whitish in color, dense to the touch. Subsequently, the color of the nodule became pinkish. The pink color is determined by the presence of pigment in the nodules, according to chemical composition close to blood hemoglobin. In this regard, the pigment is called leghemoglobin (legoglobin) - Leguminosae hemoglobin.

By the time of the formation of fruits (beans), necrosis of nodules began, they darkened and became softer to the touch. According to the literature data, necrosis in annual legumes begins during the period of mass flowering of the host plant. In our case, the later death of the nodules is due to the fact that we noted only visible processes noticeable to the eye, when they had already spread from the center of the nodule to its periphery. Old nodules are dark, flabby, soft. When cut, watery mucus protrudes from them. Scientists believe that the process of destruction of the nodule, starting with the corking of the cells of the vascular system, is facilitated by a decrease in the photosynthetic activity of the plant, dryness, or excessive humidity of the environment (soil and air).

Conclusion and conclusions.

The problem of preserving soil fertility is one of the most important, because the provision of people with food directly depends on it.

Attempts to solve it by applying chemical fertilizers also have negative consequences. It is not surprising that in countries with highly developed agriculture, usually up to 20-25% of the cultivated area is occupied by leguminous plants. At the same time, it is possible to obtain valuable food - the green mass of plants and the enrichment of the soil with nitrogen.

Unfortunately, amateur gardeners in their practice do not use such a simple and, more importantly, environmentally friendly art technique as sowing green manure - green fertilizers. You can often see how they are burned or taken out of the site. plant remains. But during the growing season, plants take nitrogen from the soil to build their body. The soil also loses nitrogen as a result of harvesting. And instead of planting peas, beans, clover, alfalfa, lupins, Robinia, gardeners apply nitrogen fertilizers to depleted soils, clogging the soil with nitrates. It is relevant that today, and not in the 19th century, the great Russian satirist writer M.E. Saltykov-Shchedrin wrote the lines: “Today, chemists and physicists are in use. Such a time has already come that they don’t go to church, but more, if I may say believe in fertilizers.”

Therefore, the educational aspect of the work associated with the need to grow plants of the legume family is also important.

1. Symbiotic nodules formed on the roots of all planted Pisum sativum pea plants.

2. Visible nodules on the roots of peas appear 22 days after sowing the seeds.

3. No noticeable differences in the formation of nodules in varieties "Oregon" and "Alpine Emerald" were found.

Nodules proliferation of root tissues, which are found on the resulting symbiotic association with nitrogen-fixing bacteria (most characteristic of members of the legume family).

In legume nodules, free atmospheric nitrogen is reduced to ammonium. Which is then assimilated, being a part of organic compounds. This produces amino acids (protein monomers), nucleotides (DNA and RNA monomers, as well as the most important energy-enriched molecule - ATP), vitamins, flavones, and phytohormones.

The ability to symbiotic nitrogen fixation of atmospheric nitrogen makes legumes an ideal crop for cultivation, due to the reduced need for nitrogen fertilizers. Moreover, the high content of nitrogen forms available for the plant (nitrate NO 3 - and ammonium NH 4 +) in the soil blocks the development of nodules, since the formation of symbiosis for the plant becomes unreasonable.

The energy for nitrogen fixation in the nodules is formed as a result of the oxidation of sugars (products of photosynthesis) coming from the leaves. Malate, as a breakdown product of sucrose, is a source of carbon for symbiotic bacteria.

The process of atmospheric nitrogen fixation is extremely sensitive to the presence of oxygen. In this connection, legume nodules contain an iron-containing oxygen-binding protein - legoglobin. Legoglobin is similar to animal myoglobin, which is used to facilitate the diffusion of oxygen used in cellular respiration.

Symbiosis

legume family

Many representatives of legumes (Fabaceae) are capable of symbiotic nitrogen fixation: pueraria, clover, soybeans, alfalfa, lupine, peanut and rooibos. In the root nodules of plants there are symbiotic rhizobia  (nodule bacteria). Rhizobia produce nitrogen compounds necessary for growth and competition with other plants. When a plant dies, the fixed nitrogen is released, making itself available to other plants, thereby enriching the soil with nitrogen. The vast majority of legumes have such formations, but some (for example, Styphnolobium) do not. In many traditional methods In agriculture, the fields are sown with different types of plants, and this change of species is cyclical. Examples of such plants include clover and buckwheat (not legumes, family Polygonaceae). They are also called "green manure".

Another agricultural method of growing agricultural plants is to plant them between rows of Inga trees. Inga is a small tropical hard-leaved tree capable of forming root nodules and, accordingly, nitrogen fixation.

Plants not in the legume family

While most plants capable of producing nitrogen-fixing root nodules today are in the legume family, there are a few exceptions:

• Parasponia is a tropical genus of the hemp family, capable of interacting with rhizobia and forming nitrogen-fixing nodules;
• Actinorizal plants, such as alder and waxwort, can also form nitrogen-fixing nodules through a symbiotic relationship with Frankia bacteria. These plants belong to 25 genera belonging to 8 families.

The ability to fix nitrogen is not ubiquitous in these families. For example, out of 122 genera in the Rosaceae family, only 4 are able to fix nitrogen. All families belong to the orders Cucurbitaceae, Beechaceae and Rosaceae, which, together with the Legumes, form a subclass of Rosida. In this taxon, the Beans were the first to branch off from it. Thus, the ability to fix nitrogen may be plesiomorphic and subsequently lost in most descendants of the original nitrogen fixing plant. However, it is possible that the main genetic and physiological prerequisites could have been present in the last universal common ancestor of all plants, but were realized only in some modern taxa.

Family: Genus Birch: Alder(alders) Cannabis: Trema loch Sea ​​buckthorn sheferdia

Comptonia Morella Mirika

red-root College Discaria Kentrothamnus Retanilla Talguenea Trevoa

Cercocarpus Chamaebatia Dryad Purshia/Cowania

Classification

On the this moment There are two main types of root nodules: deterministic and indeterminate.

Deterministic root nodules found in certain taxa of tropical legumes such as the genus Glycine (soybean), Phaseolus (beans) and Vigna, and some Lotus. Such root nodules lose their meristematic activity soon after formation, so growth is due only to an increase in cell size. This leads to the formation of mature nodules of spherical shape. Other types of deterministic root nodules are found in many herbs, shrubs, and trees (eg, peanuts). They are always associated with the axils of lateral or adventitious roots and form as a result of infection through lesions (eg, fissures) in which these roots form. Root hairs are not involved in the process. Their internal structure is different from that of soybeans. Cite error : Invalid call: key was not specified

Non-deterministic root nodules are found in most legumes of all three subfamilies both in the tropics and in temperate latitudes. They can be found in papilioinoid legumes such as Pisum (pea), Medicago (alfalfa), Trifolium (clover), and Vicia (vetch), as well as in all mimosoid legumes, such as acacia, and in caesalpinioids. These nodules are called "indeterminate" due to the fact that their apical meristem is active, which leads to the growth of the nodule throughout its life. As a result, a nodule is formed, which has a cylindrical, sometimes branched shape. Due to the fact that they are actively growing, it is possible to distinguish zones that delimit various stages of development and symbiosis:

Zone I - active meristem. Here, new nodule tissues are formed, which then differentiate into other zones.
Zone II - zone of infection. This zone is riddled with infectious threads, consisting of bacteria. Plant cells here are larger than in the previous zone, cell division stops.
Interzone II-III - entry of bacteria into plant cells containing amyloplasts. The cells elongate and begin to finally differentiate into symbiotic, nitrogen-fixing bacteria. Zone III - nitrogen fixation zone. Each cell in this zone has a large central vacuole and the cytoplasm is filled with symbiotic nitrogen-fixing bacteria. The plant fills these cells with leghemoglobin, which gives them a pink hue; Zone IV - aging zone. Here degradation of cells and their endosymbionts occurs. Destruction of the leghemoglobin heme results in a green tint. This is the most studied type of root nodule, but the details are different in the nodules of peanuts and related plants, as well as nodules of agricultural plants such as lupins. Its nodules are formed due to direct infection by rhizobia of the epidermis, where infectious threads are not formed. The nodules grow around the root, forming a ring-like structure. In these nodules, as well as in peanut nodules, the central infected tissue is homogeneous. Soybeans, peas and clover show a lack of uninfected cells in the nodules.

Formation of a root nodule

The roots of legumes secrete flavonoids, which induce the production of nod factors in bacteria. When this factor is recognized by the root, a number of morphological and biochemical changes occur: cell divisions are initiated in the root to create a nodule, and the growth trajectory of the root hair changes so that it envelops the bacterium up to its complete encapsulation. Encapsulated bacteria divide several times, forming a microcolony. From this colony, bacterial cells enter the developing nodule via a structure called an infection thread. It grows through the root hair up to the basal part of the epidermal cell, and then to the center of the root. Bacterial cells are then surrounded by the membrane of plant root cells and differentiate into bacterioids capable of fixing nitrogen.

Normal tuberization takes approximately four weeks after planting. The size and shape of the nodules depends on the type of plant that has been planted. Thus, soybeans or peanuts will have larger nodules than fodder legumes (red clover, alfalfa). By visually analyzing the number of nodules as well as their color, scientists can determine the nitrogen fixation efficiency of a plant.

Nodule formation is controlled by both external processes (heat, soil pH, drought, nitrate levels) and internal processes (autoregulation of tuberization, ethylene). Autoregulation of tuberization controls the number of nodules in a plant through processes involving leaves. Leaf tissue senses the early stages of tuberization through an unknown chemical signal and then limits further development nodule in developing root tissue. Leucine-rich repeats (LRR) of receptor kinases (NARK in soybeans (Glycine max); HAR1 in Lotus japonicas, SUNN in Medicago truncatula) are involved in the autoregulation of tuberization. Mutations leading to the loss of function of these receptor kinases lead to increased levels of tuberization. Often, root growth anomalies are accompanied by a loss of activity of the discussed receptor kinases, which indicates a functional relationship between the growth of nodules and roots. The study of the mechanisms of nodule formation showed that the ENOD40 gene, encoding a protein of 12-13 amino acids, is activated during tuberization.

Relationship with the root structure

Apparently, root nodules in representatives of the legume family were formed at least three times in the course of evolution and are rarely found outside this taxon. The tendency of these plants to develop root nodules is most likely related to root structure. In particular, the tendency to develop lateral roots in response to abscisic acid may contribute to the later evolution of root nodules.

Root nodules in other plant species

Root nodules that occur in members of other families, such as Parasponia, a symbiosis with bacteria of the genus Rhizobium, and those that result from symbiotic interactions with Actinobacteria Frankia, such as alder, differ significantly from the forms of nodules formed in legumes. In this type of symbiosis, the bacteria never emerge from the infection threads. Actinobacteria Frankia forms symbiotic relationships with the following taxa (the family is indicated in brackets): Cucurbitaceae (Coriaria and Datisca), Beechaceae (Birch, Casuarina and Cerebraceae), Rosaceae (Crushinaceae, Lochaceae and Pink). Actinorizal symbioses and rhizobial symbioses are similar in the efficiency of nitrogen fixation. All of these orders, including the Fabales, form a single nitrogen-fixing taxon with the broader Rosidae taxon.

Some fungi form tuberous structures known as tuberculate mycorrhizae on the roots of host plants. For example, Suillus tomentosus forms such structures with pine larch (Pinus contorta var. Latifolia). These structures have been shown to contain bacteria that are able to fix nitrogen. They fix a large amount of nitrogen and allow pines to colonize new territories with poor soils.

Mycorrhiza is a mutualistic (symbiotic) association between the fungus and the roots of the plant. Apparently, the vast majority of terrestrial plants enter into such relationships with soil fungi, which is of great importance, since as a result, many mineral elements and energy also enter the roots of plants. Mushrooms receive organic nutrients from plants, mainly carbohydrates and vitamins, and in return, plants receive mineral salts through their roots (mainly

mycorrhiza There are two types - ecto- and endotrophic. Ectotrophic mycorrhiza forms a sheath around the root and penetrates into the air spaces between the cells of the skin, without penetrating, however, into the cells. Thus, an extensive intercellular network is formed. It is formed by mushrooms belonging to the category of edible fibs; you can find it mainly in forest plants, such as conifers, beech, oak and many others. Fruiting bodies, in fact, the mushrooms that we collect, can often be seen near these trees.

Endotrophic mycorrhiza found in almost all other plants. Like ectotrophic mycorrhiza, it forms an intercellular network that also spreads in the soil; however, in this case, the fungi penetrate into the cells (although, in fact, the plasma membrane of the root cells remains intact).

Further study structure and functions of mycorrhiza will allow applying the acquired knowledge in agriculture and forestry and in carrying out land reclamation work.

root nodules

Nitrogen fixation by root nodules of leguminous plants is discussed in our article. Bacteria inhabit the nodules, which stimulate the growth and division of root parenchymal cells, resulting in the formation of swellings, or nodules, on the roots.

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

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

Rice. 168. Alder nodule tissue (according to J. Becking).

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

Rice. 169. Nodules on tribulus roots (according to E. and O. Allen).

In a number of representatives of angiosperms, 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 semishrubs (families Coriariaceae, Myricaceae, Betulaceae, Casuarinaceae, Elaeagnaceae, Rhamnaceae) included in this group. The nodules of most representatives of this group are coral-like clusters of pink-red color, acquiring a brown color with age. There is evidence of the presence of hemoglobin in them. In species of the genus Elaeagnus (loch) nodules are white.

Rice. 170. Nodules on the roots of the forest weisht (according to I.L. Klevenskaya).

Often nodules are large. In casuarina (Casuarina) they reach a length of 15 cm. They function for several years.

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

Many plants of the group under consideration grow on nutrient-poor soils - sands, dunes, rocks, swamps.

The nodules of alder (Alnus), in particular A. glutinosa, discovered in the 70s of the last century by M. S. Voronin, have been studied in the most detail (Fig. 167). There is an assumption that nodules are characteristic not only of modern, but also of 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.

Rice. 171. Scheme of the structure of the nodule of the forest reed grass: 1 - bark, 2 - bark parenchyma; h - pericyclic parenchyma; 4 - vascular bundle; 5 - endoderm; 6 - bacteria (according to I. L. Klevenskaya).

Endophyte in nodules is polymorphic. It usually occurs as hyphae, vesicles, and bacteroids (Fig. 168). The taxonomic position of the endophyte has not yet been established, since numerous attempts to isolate it into a pure culture turned out to be fruitless, and if it was possible to isolate the cultures, they 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 the endophyte. Growing in areas where the cultivation of agricultural plants is not economically rational, they play the role of pioneers in the development of the land. Thus, the annual increase in nitrogen in the soil of the dunes of Ireland (Cape Verde) under plantings of Casuarina equi-setifolia reaches 140 kg/ha. The content of nitrogen in the soil under alder is 30-50% higher than under birch, pine, and willow. In the dried leaves of alder, nitrogen is twice as much as in the leaves of other woody plants. According to the calculations of A. Virtanen (1962), an alder grove (an average of 5 plants per 1 m 2) gives an increase in nitrogen of 700 kg/ha in 7 years.

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

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

It is interesting to note that tropical plants such as the bright red parophyllum form nodules only at high temperatures and low soil moisture. Soil moisture up to 80% of the total moisture capacity prevents the formation of nodules. As is known, the reverse phenomenon is observed in leguminous plants of a temperate climate. With insufficient moisture, they do not form nodules.

The nodules in plants of the Parnolistaceae family differ 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 found on lateral roots and at greater depths. Sometimes nodules form on stems if they lie on the soil surface.

The nodules of terrestrial 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 bark.

In the bright red parnolistnik, the nodules are the terminal thickenings of the lateral roots of plants. Bacteroids are found in nodules; bacteria are very similar to root nodules.

Nodules of tropical plants Tribulus cistoides are hard, rounded, about 1 mm in diameter, connected to the roots by a wide base, often whorled on old roots. More often 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 cell division of the pericycle of the stele.

Histological study of nodules of Tribulus cistoides 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.

The nodules of the forest reedweed are spherical or somewhat elongated formations up to 4 mm in diameter, tightly seated on the roots of plants (Fig. 170). The color of young nodules is most often white, occasionally pinkish, old -yellow and brown. The nodule is connected with the central cylinder by a wide vascular bundle. Just like in Tribulus cistoides, reed nodules have bark, bark parenchyma, endoderm, pericyclic parenchyma and vascular bundle (Fig. 171).

Bacteria in nodules of wood reedweed are very reminiscent of root nodule bacteria of leguminous plants.

Nodules are 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 coffee plants Coffea robusta and Goffea klainii. They branch dichotomously, sometimes flattened and look like a fan. Bacteria and bacteroid cells are found in the tissues of the nodule. Bacteria, according to Steyart (1932), belong to Rhizobium, but they named them Bacillus coffeicola.

Nodules in plants of the rose family were found on the dryad (partridge grass). Two other members of this family, Purshia tridentata and Cercocarpus betuloides, have described typical coral nodules. However, there are no data on the structure of these nodules and the nature of their pathogen in the literature.

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

In angiosperms monocotyledonous plants, nodules are common among representatives of the cereal family: meadow foxtail, meadow bluegrass, Siberian hairweed and saline hairweed. Nodules are formed at the ends of the roots; are oblong, rounded, fusiform. In the foxtail, young nodules are light, transparent or translucent, becoming brown or black with age. Data on the presence of bacteria in nodule cells are contradictory.

Leaf nodules. Over 400 species of various plants are known to form nodules on leaves. The nodules of Pavetta and Psychotria have been studied the most. They are located on the lower surface of the leaves along the main vein or are scattered between the lateral veins, have an intense green color. Chloroplasts and tannin are concentrated in 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 growing sterile seeds, nodules do not appear and the plants develop chlorotic. Bacteria isolated from the leaf nodules of Psychotria bacteriophyla 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 microorganisms of the phyllosphere - a special kind of epiphytic microorganisms, which are also involved in the nitrogen nutrition of plants. The bacteria of the phyllosphere are predominantly oligonitrophils (they live on negligible impurities of nitrogen-containing compounds in the medium and, as a rule, fix small amounts of molecular nitrogen), which are in close contact with the plant.

Free-living nitrogen-fixing microorganisms. Azotobacter (AZOTOBACTER)

In 1901, Beijerinck isolated an aerobic, non-spore-forming gram-negative bacterium that fixes molecular nitrogen from the soil and named it Azotobacter chroococcum (the generic name reflects the ability of the bacterium to fix nitrogen, while the species name reflects the ability to synthesize a brown pigment - chroo and form coccoid cells - coccum). Azotobacter is a typical representative of free-living microorganisms. Free-living - these are all those microorganisms that live in the soil, regardless of whether the plant develops near or not.

Rice. 173. Dividing cells of Azotobacter (A. agilis), peritrichial flagella are visible (7), in A. macrocyto-genes polar flagella (?) are visible (according to A. Bayle et al.) -

Azotobacter cultures in the laboratory are characterized by polymorphism. Cells of various Azotobacter species in young age shown in Figure 172. Young Azotobacter cells are mobile; they have numerous or single flagella (Fig. 173). Azotobacter has pili-like outgrowths (Fig. 174). In old cultures, Azotobacter cells are covered with a dense membrane, forming cysts. They can germinate, giving rise to young cells (Fig. 175).

Azotobacter polymorphism depends to a large extent on the composition of the medium on which it is grown. On a medium with ethyl alcohol (as the sole source of carbon), Azotobacter retains its mobility and rod shape for a long time. At the same time, polymorphism manifests itself very sharply in many other environments.

Rice. 174. Fimbra-like formations in Azotobacter cells. Increased X 24,000. (According to E. V. Boltyanskaya.)

On dense nutrient media that do not contain nitrogen, Azotobacter forms large slimy, sometimes wrinkled colonies (Fig. 176), which turn yellowish-greenish, pink or brown-black with aging. Colonies of different species of Azotobacter have their own specific pigmentation.

To date, a number of species of Azotobacter are known: Azotobacter chroococcum, Az. beijerinckii, Az. vinelandii, Az. agilis, Az. nigricans, Az. galophilum.

Rice. 175. Azotobacter cysts (according to I. Chan and others). A mature cyst filled with fat granules and surrounded by a thick hard shell (right) and a germinating cyst (a growing young cell ruptures the cyst shell - left). Increased X 35,000.

A variety of mineral (ammonium salts, nitric and nitrous acids) and organic (urea, various amino acids) compounds can serve as a source of nitrogen for Azotobacter. However, if Azotobacter develops only at the expense of nitrogen bound in the environment, it does not fulfill its main function - the fixation of molecular nitrogen. Azotobacter usually fixes up to 10-15 mg of molecular nitrogen per 1 g of carbon source used (eg glucose, sucrose). This value varies greatly depending on the growing conditions of the culture, the composition of the nutrient medium, its acidity, temperature, and aeration.

In relation to carbon sources, VL Omelyansky (1923) called Azotobacter a polyphage (“omnivore”).

Rice. 176. Development of A. chroococcum colonies around soil lumps in a nitrogen-free environment.

Azotobacter assimilates a variety of carbohydrates (mono- and disaccharides, some polysaccharides), organic acids, polyhydric alcohols (glycerol, mannitol) and other substances well.

Many researchers have been able to grow Azotobacter in plates with a nutrient medium without nitrogen and carbon, if the plates were placed in a chamber containing vapors of acetone, ethyl alcohol, or some other organic compounds. In the presence of readily available forms of carbon-containing compounds, Azotobacter can partially use carbon dioxide from the atmosphere. Increasing the concentration of carbon dioxide to 0.5% in the air somewhat stimulates the development of Azotobacter. But easily accessible forms of carbon-containing organic compounds are better absorbed by Azotobacter. In the soil, the reserve of mobile organic matter is small, therefore, it is the lack of readily available carbon compounds that primarily limits the development of azotobacter in natural conditions.

Rice. 177. Beyerinkia colonies of different types (smooth and folded variants): 1-4, 6-8, 10 - according to N. I. Gogorikidze; 5, 9, 11 - according to J. Becking.

What organic compounds can Azotobacter use in the soil? The humus substances of the soil are practically not absorbed by Azotobacter. Therefore, in soils, even very rich in humus, in the absence of fresh organic residues, intensive reproduction of Azotobacter does not occur.

However, if there are organic compounds and decay products of plant and animal cells in the soil, Azotobacter develops well. In particular, it multiplies intensively in soils fertilized with straw and straw manure, as well as in various composts containing cellulose. Azotobacter well assimilates substances formed during the breakdown of cellulose.

The development of Azotobacter and its fixation of nitrogen largely depend on the presence of phosphorus in the environment. Both organic and mineral phosphorus-containing compounds can serve as a source of phosphorus. The high sensitivity of Azotobacter to phosphorus made it possible to develop a microbiological method for determining the need for soils in phosphate fertilizers.

Azotobacter is used as a test organism in this method. Microbiological methods for determining the need for soil fertilizer have a number of advantages over chemical analyzes, although, of course, they are inferior in accuracy.

Calcium plays an important role in the metabolism of Azotobacter. This element is necessary for Azotobacter when fed with both molecular and ammonium nitrogen (G.N. Zaitseva, 1965). The lack of calcium in the medium leads to strong vacuolization of cells and their swelling.

The high sensitivity of Azotobacter to calcium, as well as to phosphorus, is used to determine the need for liming in soils.

Trace elements (molybdenum, boron, vanadium, iron, manganese) are necessary for Azotobacter primarily for the implementation of the process of nitrogen fixation. The need for trace elements is determined to a large extent by the geochemical conditions for the existence of Azotobacter in soils. Microorganism strains isolated from soils with a high natural content of one or another microelement require, as a rule, higher concentrations of these elements.

Interestingly, radioactive elements (radium, thorium, uranium) have a stimulating effect on the development of Azotobacter and the process of nitrogen fixation.

Azotobacter is extremely sensitive to environmental reactions. The optimal pH range for its development is 7.2-8.2. However, Azotobacter is able to develop on media with a pH of 4.5 to 9.0; the acid reaction of the environment adversely affects its development. From acidic soils, inactive forms of Azotobacter are isolated, which have lost the ability to bind molecular nitrogen.

Soil moisture has a great influence on the development of Azotobacter. Azotobacter cells have a lower osmotic pressure than the cells of fungi and actinomycetes; the need for moisture is similar to the need for higher plants. Azotobacter is common in fresh water bodies, silts, flooded rice fields, sewage, highly moist soils, on aquatic plants in ponds and reservoirs. This indicates its high degree of hydrophilicity. Based on the high moisture requirement of soil forms of Azotobacter, it is assumed that the ancestors of some marine and soil species of Azotobacter could be common.

With regard to temperature, Azotobacter is a typical mesophilic organism, with an optimum development of about 25-30 °C. Azotobacter tolerates a decrease in temperature well, therefore, even in northern latitudes, the number of its cells in the soil does not noticeably decrease in winter.

Of the biological factors influencing the development of Azotobacter, soil microorganisms should be noted first of all. They can influence the vital activity of Azotobacter in the soil indirectly by changing, for example, pH or redox conditions, and directly by producing nutrients and biologically active substances. Thus, the activating effect of cellulose-destroying and butyric microorganisms on the development of Azotobacter and its antagonistic relationship with representatives of the soil microflora was noted by many Soviet and foreign researchers. The biocenosis of microorganisms, which is formed under the conditions of a particular soil, changes to a large extent under the influence of the vegetation cover. And Azotobacter as a member of the biocenosis also depends on this factor. It has been established using the method of autoradiography that when phosphorus-labeled Azotobacter cells are applied to seeds of cereal crops, the cells usually concentrate around the growing root system of seedlings.

There is, however, evidence that there are very few Azotobacter cells in the plant rhizosphere. In the best case (with the complete absence of antagonists and favorable environmental conditions), their number does not exceed 1% of total number rhizosphere microflora.

Azotobacter cultures, as a rule, form a significant amount of biologically active substances: B vitamins, nicotinic and pantothenic acids, biotin, heteroauxin and gibberellin. However, despite the fact that cultures of Azotobacter produce a whole series of biologically active substances, the addition of vitamins, gibberellin and heteroauxin to the medium accelerates the growth of Azotobacter. The reaction to the additional introduction of vitamins into the medium is an individual feature of the strains.

Azotobacter can produce growth substances such as auxins. This is confirmed by experiments in which the formation of additional roots in bean cuttings under the influence of auxins produced by Azotobacter was established. A biological test - a dwarf form of pea variety Pioneer - allows you to determine gibberellin-like compounds in azotobacter culture.

All these compounds together are capable of stimulating the germination of plant seeds and accelerating their growth in those cases, of course, when there are a sufficient number of Azotobacter cells on the plant root system.

In addition, the antagonistic activity of Azotobacter against pathogens of bacterial plant diseases was found. Azotobacter synthesizes a fungistatic (delaying the development of fungi) antibiotic of the anisomycin group. A number of fungal organisms found on seeds and in soil (species from the genera Fusarium, Alternaria, Penicillium) can inhibit the development of many plant species, especially in cold weather. Azotobacter, by producing antifungal antibiotic substances, helps plants grow and develop, which is especially important in the early phases of development.

Unfortunately, the ability of azotobacteria to propagate in the soil and to show multifaceted qualities is very limited due to the lack of readily available organic substances in the soil and the high demands of the world organism on environmental conditions. Therefore, the stimulating effect of Azotobacter is manifested only on fertile soils.

The distribution of Azotobacter in the soils of the Soviet Union has certain regularities. In virgin podzols and soddy-podzolic soils characterized by an acidic reaction, the conditions for the development of Azotobacter are unfavorable. Only the cultivation of such soils creates opportunities for its development. In soils with increased moisture and a predominance of meadow vegetation (floodplain soils), Azotobacter is usually found throughout the growing season in large quantities. In peatlands Azotobacter is either absent or develops very weakly. Azotobacter develops well in the zone of sufficiently moist northern thick chernozems, and in the zone of ordinary and southern chernozems in the absence of irrigation, as well as in virgin and non-irrigated cultivated chestnut soils only as a spring ephemeral. The maximum development of Azotobacter in the spring period is observed both in virgin and rainfed soils of the serozem zone. Predominantly salt-resistant races of Azotobacter are common in solonetzes and solonchaks. Basically, Az dominates in the soils of our country. chroococcum.

Beyerinkia (BEIJERINCKIA)

For the first time, aerobic bacteria of the genus Beijerinckia were isolated from the acidic soils of rice fields in India (in 1939). G. Derks (1950), having discovered this bacterium in the soil of the Botanical Garden in Bogor (Java), proposed to name it after M. Beijerinck, one of the first researchers of nitrogen fixers.

Cells of bacteria of the genus Beijerinckia are round, oval or rod-shaped; sticks are sometimes curved. The size of young cells is 0.5-2.0 X 1.0-4.5 microns. There are mobile and fixed forms. Cysts and spores do not form. Cultures are characterized by slow growth. Typical colonies usually form after 3 weeks at 30°C. Most cultures of Beijerinckia form convex, often folded, shiny mucous colonies of a very viscous consistency on nitrogen-free agar with glucose (Fig. 177). As cultures age, they tend to form a dark-colored pigment.

Organisms of the genus Beijerinckia fix 16-20 mg of molecular nitrogen per 1 g of used energy material. The range of carbon-containing compounds available to Beyerinkia is much narrower than that of Azotobacter. Mono- and disaccharides are well used, worse - starch, organic acids, aromatic substances are not absorbed. Bacteria of the genus Beijerinckia prefer mineral nitrogen and many amino acids to molecular nitrogen.

The main differences between Beyerinkia and Azotobacter are high acid resistance (they can grow even at pH 3.0), calcephobicity (negligible doses of calcium inhibit growth), and resistance to high concentrations of iron and aluminum.

Bacteria of the genus Beijerinckia are widespread in the soils of the southern and tropical zones, and are less common in the temperate zone. Beijerinckia is often found on the leaf surface of tropical plants in Indonesia.

Previously, it was believed that bacteria of the genus Beijerinckia could only exist in acidic soils. It has now been established that they develop well in neutral and alkaline soils. Nevertheless, it should be assumed that Beijerinckia play a significant role in the nitrogen balance of mainly acidic soils (laterites, krasnozems), having no significant agronomic significance for neutral soils.

Clostridium (CLOSTRIDIUM)

The first anaerobic microorganism that absorbs molecular nitrogen was isolated and described by S. N. Vinogradsky in 1893. It turned out to be a spore-forming bacterium, which was given the name Clostridium pasteurianum (the generic name comes from the Latin word clostrum - spindle; specific - pasteurianum - given in honor Louis Pasteur).

Cells Cl. pasteurianum are large, their length is 2.5-7.5 microns, their width is 0.7-1.3 microns. They are located singly, in pairs or form short chains. Young cells are motile, have peritrichous flagella, their plasma is homogeneous. As the cell ages, the plasma becomes granular and accumulates granulosa (a starch-like substance). In the center of the cell or closer to its end, a spore is formed, which is much wider in diameter than the vegetative cell, and therefore the cell takes the form of a spindle during this period. The spore size is 1.3 x 1.6 µm. Figure 178 shows the cells of Cl. pasteurianum with spores. The morphology of spores and the behavior of the nuclear substance in the process of spore formation in Clostridium are described in detail on p. 228 by V. I. Duda.

Rice. 178. Cells of Clostridium pasteurianum with spores. Increased X 3500 (according to V.I. Duda).

The nitrogen-fixing function was found in many representatives of the genus Clostridium: Cl. pasteurianum, Cl. butyricum, Cl. butylicum, Cl. beijerinckia, Cl. pectinovorum, Cl. acetobutyli-cum and other species. The most energetic nitrogen accumulator is Cl. pasteurianum - fixes 5-10 mg of nitrogen per 1 g of carbon source consumed.

Along with molecular nitrogen, bacteria of the genus Clostridium well assimilate mineral and organic nitrogen-containing compounds. Bacteria of the genus Clostridium use various compounds as a source of carbon nutrition, which usually serve as an energy source for them at the same time. They are much less sensitive to phosphorus, potassium and calcium than Azotobacter. However, soil fertilization with phosphorus-potassium salts, liming of soils or composts always leads to an increase in abundance.

Clostridia are relatively resistant to acid and alkaline reaction environment. The pH range at which their development proceeds normally is quite wide; the minimum pH value is below 4.5, the maximum is above 8.5.

The influence of the air-water regime on the development of bacteria of the genus Clostridium has been studied quite fully. Being anaerobic, they tolerate high soil moisture saturation well. However, the optimal degree of moisture for them is determined by the type of soil and the availability of organic matter. Clostridia develops best when the soil moisture is 60-80% of the total moisture capacity.

Table 46. Bacteriosis of oats and Sudan grass: 1 - brown (red) bacteriosis of oats; 2 - bacteriosis of Sudan grass.

Most bacteria of the genus Clostridium are in the upper layers of the soil, which are rich in organic matter.

Bacteria of the genus Clostridium but differently related to temperature, are found as mesophilic. and thermophilic bacteria. Molecular nitrogen is fixed only by mesophiles.

In mesophilic forms, the optimal development temperature is most often in the range of 25-30 °C. The maximum temperature limit is 37-45 °C.

Table 47. Diseases of cotton, tobacco and beets: 1 - cotton blight; 2 - bacterial grouse tobacco; 3 - silver beet disease (on the right - spots under magnification).

Clostridial spores are very resistant to high temperatures. They withstand heating at 75 °C for 5 hours and for 1 hour heating at 80 °C. The spores of thermophilic clostridia die when boiled after 30 mcn. Higher temperatures (110°C) quickly kill them.

With many microorganisms in the soil, Clostridium is in a metabolic relationship, in which the exchange of metabolic products is expected. Thus, Azotobacter improves the living conditions of Clostridium by absorbing oxygen, and Clostridium produces organic acids from organic compounds that are inaccessible to Azotobacter, which Azotobacter can assimilate.

It would be difficult to answer the question: what soils do not have Clostridium? "Omnivorous" Clostridium, low exactingness to the conditions external environment, as well as the ability to go into a state of dispute under adverse conditions, explain their wide, almost ubiquitous distribution.

The accumulation of nitrogen in soils due to the activity of Clostridium, however, is small and, as a rule, does not exceed a few kilograms per hectare of soil.