Bacteria and Archaea of the Agrosphere

1.1. Prokaryotes: definition and place in the system of the organic world

To begin, let us recall the key definition. Prokaryotes (from Greek pro — before, karyon — nucleus) are organisms whose cells lack a true, membrane-bound nucleus. Their genetic material (DNA) is not separated from the cytoplasm by a nuclear envelope and is usually represented by a single circular DNA molecule located in the nucleoid (Weil, 2017; Scheffer et al., 2018).

In this they fundamentally differ from eukaryotes (plants, animals, fungi), whose cells contain true organelles, including a nucleus, mitochondria, and, in plants, chloroplasts. In the system of the organic world, prokaryotes occupy a separate position, representing two independent branches of evolution — the domains Bacteria and Archaea.

What is their "invisible foundation" for the agrosphere? The answer lies in their colossal abundance and ubiquitous distribution. If we take just one gram of fertile soil, we will find from several billion to hundreds of billions of bacterial cells in it (Weil, 2017). Their biomass in the upper soil layer can reach several tons per hectare (about 400–5000 kg/ha in live weight), which is comparable to the biomass of a large herd of animals (Weil, 2017). These figures allow us to say that it is prokaryotes, along with fungi, that are the main "conductors" of soil processes.

1.2. Functional role of prokaryotes in agroecosystems

What work do these invisible laborers perform? Their functions cannot be overestimated. In the context of our discipline, where we consider organisms as part of an agroecosystem, the role of prokaryotes comes down to several key areas.

First, the cycling of biogenic elements. Prokaryotes are the main mediators in the global cycles of carbon, nitrogen, phosphorus, and sulfur. They decompose organic matter, returning forms of elements available to plants to the soil. Without their activity, soil fertility would be impossible, since the main nutrients would be "locked up" in dead organic matter (Huang et al., 2012; Scheffer et al., 2018).

Second, direct influence on fertility and plant health. Bacteria and archaea do not merely mineralize organic matter. They enter into complex, and sometimes symbiotic, relationships with plants. Some fix atmospheric nitrogen, making it available to plants (Marschner, 2012), others dissolve sparingly soluble phosphates, and still others produce phytohormones that stimulate growth (Glick, 2012; Lugtenberg & Kamilova, 2009). There are also those that protect plants from pathogens, acting as biocontrol agents (Compant et al., 2005).

Third, diversity that boggles the imagination. Among prokaryotes we find the full spectrum of relationships with plants: from ruthless pathogens that cause diseases and reduce yields (Agrios, 2005) to faithful symbionts and beneficial associative partners. This diversity is the guarantee of stability and resilience of the agroecosystem. Understanding this diversity is the key to managing soil health and plant productivity.

1.3. Diversity of prokaryotes: from pathogens to symbionts

Let us dwell on this spectrum in a little more detail. Traditionally, when we think of bacteria in agriculture, we first think of plant diseases. Indeed, pathogens such as Pseudomonas syringae, Xanthomonas campestris, or Ralstonia solanacearum cause enormous damage to agriculture worldwide (Agrios, 2005). We will study them in detail in the relevant sections.

However, we should not forget that pathogens are only the tip of the iceberg. A much larger and more important group for agronomy consists of beneficial microorganisms. These include:

• Symbiotic nitrogen fixers: for example, nodule bacteria of the genus Rhizobium, which enter into a close union with legumes, providing them with nitrogen (Udvardi & Poole, 2013).
• Associative and free-living nitrogen fixers: such as Azospirillum and Azotobacter, which live in the rhizosphere (root zone) and also supply plants with nitrogen (Glick, 2012).
• Phosphate-mobilizing bacteria: for example, Bacillus and Pseudomonas, capable of converting insoluble phosphates into a plant-available form.
• Biocontrol agents: for example, Bacillus subtilis or Pseudomonas fluorescens, which suppress the growth of phytopathogens, protecting the crop (Lugtenberg & Kamilova, 2009).
• Plant growth-promoting rhizobacteria (PGPB): a broad group that directly or indirectly improves plant growth and development (Compant et al., 2005; Glick, 2012).

It is precisely this diversity — from pathogens to symbionts — that constitutes the subject of our further study. We will see that the systematics and taxonomy of these organisms is not merely an academic game of the mind, but a necessary tool for understanding their ecology and practical use.

2.1. The three-domain system: Carl Woese's revolution

For a long time, biology was dominated by a two-kingdom or, later, five-kingdom system, in which all prokaryotes were considered a single kingdom Monera (or simply "bacteria"). They were contrasted with eukaryotes, which were divided into animals, plants, fungi, and protists. However, this system was built on morphological and physiological characters that are very limited in microorganisms and often do not reflect their true kinship.

The revolution occurred in 1977, when the American microbiologist Carl Woese and his colleague George Fox proposed a completely new approach. They decided to compare not external characters, but sequences of ribosomal RNA (rRNA), in particular the small subunit (16S rRNA in prokaryotes and 18S in eukaryotes). This choice was not accidental: rRNA is present in all living organisms, performs a vital function, and, most importantly, its sequence changes very slowly. This makes it an ideal "molecular chronometer" for measuring evolutionary distances (Doolittle & Papke, 2006).

The results were stunning. Woese discovered that the so-called "bacteria" actually divide into two groups that differ from each other no less than each of them differs from eukaryotes. He named one group Bacteria (true bacteria), and the other Archaea (from Greek archaios — ancient). Together with eukaryotes, they formed three fundamental domains of life — the highest taxonomic rank. This three-domain system (Woese et al., 1990) is generally accepted today, although its details continue to be refined.

2.2. Fundamental differences between Bacteria and Archaea

So what distinguishes these two domains? The differences are so profound that they can be found at all levels of cell organization: from membrane structure to the operation of the genetic apparatus.

1. Cell wall. In most bacteria, the cell wall contains peptidoglycan (murein) — a complex polymer consisting of carbohydrate chains cross-linked by short peptides. It is this that gives the cell its shape and protects it from osmotic pressure. Archaea lack peptidoglycan. Their cell walls are built from other polymers, often pseudopeptidoglycan (in methanogens), or they may be represented by protein or polysaccharide layers. This is a fundamental difference (Weil, 2017; Scheffer et al., 2018).

2. Membrane lipids. This is perhaps one of the most reliable differences. The membranes of bacteria and eukaryotes are built on complex esters with fatty acid residues. The membranes of archaea consist of simple ethers linked to branched hydrocarbon chains (isoprenoids). This chemical difference makes archaeal membranes more resistant to extreme conditions (high temperatures, acidity), which explains why many archaea are extremophiles (Weil, 2017).

3. RNA polymerases. The enzyme responsible for RNA synthesis (transcription) also differs in bacteria and archaea. The RNA polymerase of archaea is much closer in structure and composition to the eukaryotic one than to the bacterial one. This is one of the key arguments in favor of archaea and eukaryotes having a common ancestor (Weil, 2017).

4. Metabolism. Although metabolic diversity is enormous in both domains, there are also unique features. For example, the ability for methanogenesis (methane formation) is a unique feature of archaea, not found in bacteria. On the other hand, oxygenic photosynthesis (with oxygen release) is a unique feature of cyanobacteria, belonging to the domain Bacteria (Offre et al., 2013; Conrad, 2007).

These and other differences (for example, in ribosome structure, sensitivity to antibiotics) clearly delimit the two domains.

2.3. Horizontal gene transfer and its influence on phylogeny

However, as we have already mentioned, the picture is not so simple. The classical evolutionary tree, where genes are transmitted only "vertically" from ancestor to descendant, does not always work for prokaryotes. A huge role is played by horizontal gene transfer (HGT). This is the process by which an organism receives genetic material not from a parent, but from another, often unrelated, organism.

HGT can occur by several routes: through transformation (uptake of "naked" DNA from the environment), conjugation (transfer of DNA upon direct cell contact through special bridges), and transduction (transfer of DNA with the help of bacteriophages — viruses that infect bacteria) (Doolittle & Papke, 2006).

What does this mean for systematics? It means that prokaryotic genomes are mosaic. Some genes they inherited from a common ancestor (this is the "core" of the genome, responsible for fundamental processes), while others were acquired from completely different organisms. For example, antibiotic resistance genes or genes responsible for assimilation of some unusual substrate are often transmitted horizontally (Doolittle & Papke, 2006; Heuer et al., 2008).

Because of this, constructing a single and consistent phylogenetic tree based on one gene (even 16S rRNA) can be difficult. Trees built from different genes may not coincide. This has led to the understanding that prokaryotic evolution is not only a "tree" but also a "network" (Doolittle & Papke, 2006). To construct a more reliable phylogeny, modern scientists use not one but many genes (concatenated sequences) or even whole genomes.

2.4. Modern databases for taxonomy and phylogeny

Due to the avalanche-like growth in the number of sequenced genomes, the work of a systematist today is unthinkable without powerful bioinformatic resources. It is impossible to compare millions of sequences manually. Therefore, specialized databases have been created that accumulate, curate, and provide researchers with information on rRNA and genome sequences.

Among the most important for our discipline are:

• SILVA (from Latin silva — forest): This is one of the most authoritative and complete databases containing aligned rRNA gene sequences (both 16S/18S and 23S/28S) for all three domains of life. SILVA provides not only the sequences themselves but also tools for their analysis, taxonomic classification, and is constantly updated (Quast et al., 2012). For us, it is important that it allows accurate identification of microorganisms and study of their diversity.

• EzBioCloud: This is an integrated database that combines taxonomic hierarchy, 16S rRNA sequences, and whole-genome assemblies for Bacteria and Archaea. Its key feature is the use of genomic data (such as ANI — Average Nucleotide Identity) for accurate identification and delimitation of species. This database is especially useful for identifying new isolates and understanding their phylogenetic position (Yoon et al., 2017).

• GTDB (Genome Taxonomy Database): This is a database that builds taxonomy exclusively on the basis of comparison of complete genomes, using conserved protein-coding genes. It offers a standardized and, possibly, more objective taxonomic system, which often differs from the traditional one based on 16S rRNA, and allows deep evolutionary relationships to be revealed (Hug et al., 2016; Parks et al., 2018).

These databases are not merely reference books. They are working tools that allow us to ask questions: "To which species does this isolate belong?", "What is its evolutionary relationship to known pathogens?", "Which genes did it acquire as a result of HGT?" Without them, modern systematics and taxonomy are unthinkable.

3.1. 16S rRNA — the "gold standard" of molecular phylogeny

As we already mentioned in the previous lecture, the revolution in prokaryotic systematics occurred thanks to the use of the 16S ribosomal RNA (16S rRNA) gene (Woese & Fox, 1977). Why did this particular gene become the main phylogenetic marker? There are several reasons:

1. Universality. The 16S rRNA gene is present in all bacteria and archaea. It encodes the small ribosomal subunit, which is necessary for protein synthesis — a fundamental process for any cell.

2. Conservation and variability. This gene consists of alternating regions: conserved ones, which are almost identical in all prokaryotes, and variable ones, which differ among taxa. Conserved regions allow the design of universal primers for amplifying the gene in any organism, while variable regions allow its use for phylogenetic analysis and identification (Weil, 2017).

3. Absence of horizontal transfer. Unlike many other genes, 16S rRNA is not subject to horizontal transfer. This means that its evolution proceeds strictly vertically, from ancestor to descendant. This makes it a reliable "molecular chronometer" (Doolittle & Papke, 2006).

For decades, comparison of 16S rRNA sequences was the main method for constructing phylogenetic trees and determining the taxonomic position of microorganisms. Databases such as SILVA and EzBioCloud contain millions of 16S rRNA sequences, allowing rapid identification of an unknown isolate simply by comparing its sequence with known ones (Quast et al., 2012; Yoon et al., 2017).

3.2. Problems and limitations of single-gene analysis

However, as data accumulated, it became obvious that analysis of a single gene, even one as reliable as 16S rRNA, has serious limitations (Doolittle & Papke, 2006; Hug et al., 2016).

First, low resolution at the species level. The 16S rRNA gene is too conserved to reliably distinguish closely related species. Two different species may have identical 16S rRNA sequences, or their differences may be so small that they do not allow a clear boundary to be drawn. This is especially relevant for recently diverged taxa (Doolittle & Papke, 2006).

Second, genome mosaicism. As we discussed, prokaryotic genomes are mosaic due to HGT. A tree built from one gene will reflect the evolution of only that gene, not the whole organism. Trees built from different genes may have different topologies. This phenomenon is called gene tree discordance (Doolittle & Papke, 2006).

Third, the presence of plasmids and phages. Plasmids are extrachromosomal circular DNA molecules that often carry genes useful for survival under difficult conditions (for example, antibiotic resistance genes or genes for the ability to degrade pollutants). Plasmids can be transmitted horizontally, and their genes do not reflect the history of the main genome. Similarly, phages (bacterial viruses) can integrate into the genome and introduce their genes. All this further confuses the phylogenetic picture (Doolittle & Papke, 2006).

3.3. Concatenated genes (MLSA) and whole-genome methods

To overcome the limitations of single-gene analysis, more powerful approaches were developed.

MLSA (Multilocus Sequence Analysis). This is a method in which not one but several (usually 5–7) conserved "housekeeping" genes that are not subject to HGT are used to build a tree. The sequences of these genes are concatenated (combined into one long sequence), and the tree is built from this combined dataset. This significantly increases resolution and reliability of the phylogeny (Hug et al., 2016).

Whole-genome methods (Whole-Genome Sequencing). With the development of sequencing technologies, it became possible to compare not individual genes but complete genomes of organisms. The most modern approach is to build a phylogeny based on concatenated sequences of conserved protein-coding genes (for example, ribosomal proteins). This method is used in the GTDB (Genome Taxonomy Database). It allows reconstruction of evolutionary relationships with much higher resolution than 16S rRNA analysis, and often reveals completely unexpected relationships (Hug et al., 2016; Parks et al., 2018).

3.4. The concept of "species" in prokaryotes: from DNA-DNA hybridization to ANI

Now we have approached perhaps the most complex and debatable question in prokaryotic systematics: what is a species?

In eukaryotes (animals, plants), a species is often defined through the ability to interbreed and produce fertile offspring (biological species concept). But prokaryotes do not have sexual reproduction, so this concept is inapplicable (Doolittle & Papke, 2006; Yoon et al., 2017).

For a long time, DNA-DNA hybridization (DDH) was used to delimit species. This is a labor-intensive and poorly reproducible method that measured the degree of genome similarity between two strains. If similarity was above 70%, they were assigned to one species; if below, to different species. However, this method was far from ideal and did not allow the creation of a universal database.

With the advent of genomics, DDH was replaced by a much more accurate and standardized indicator — ANI (Average Nucleotide Identity). ANI is calculated by pairwise comparison of all homologous genes in two genomes. Based on numerous studies, the species threshold was established: ANI ≥ 95–96%. This means that if two strains have ANI of 95% or higher, they belong to the same species. If ANI is below 95%, they are different species (Yoon et al., 2017; Richter & Rosselló-Móra, 2009).

This threshold has become the "gold standard" of modern prokaryotic taxonomy. The EzBioCloud database actively uses ANI for identification and validation of new species (Yoon et al., 2017).

3.5. Modern databases and software

As we have already said, the work of a modern systematist is unthinkable without powerful bioinformatic resources. Let us recall the key ones in the context of phylogeny construction:

• SILVA: Provides rRNA sequence alignments, which are necessary for building trees based on 16S rRNA (Quast et al., 2012).
• EzBioCloud: Offers tools for identification based on 16S rRNA and ANI, and also contains curated taxonomy (Yoon et al., 2017).
• GTDB: Provides standardized taxonomy based on whole-genome data and phylogenetic trees built from concatenated proteins. This is the most modern and comprehensive resource for understanding prokaryotic evolution (Parks et al., 2018).

There are also many programs for phylogenetic analysis, such as RAxML, MEGA, IQ-TREE, which allow trees to be built based on various models of evolution.

4.1. Pseudomonadota (Proteobacteria) — the most diverse phylum

Historically, this group has been known as Proteobacteria. However, in modern taxonomy based on genomic data, it is often called Pseudomonadota. This is undoubtedly the largest and most diverse phylum of bacteria. Its representatives demonstrate colossal metabolic diversity: from photosynthesis to nitrogen fixation, from pathogenesis to symbiosis.

Classically, proteobacteria were divided into five classes named after letters of the Greek alphabet: Alpha-, Beta-, Gamma-, Delta-, and Epsilonproteobacteria. This division is still convenient for orientation, although it is not strictly phylogenetic.

Alphaproteobacteria

This class includes many organisms critically important for agriculture.

• Rhizobiales (Rhizobia): This is perhaps the most well-known group. It includes nodule bacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium, and others. They form symbiosis with leguminous plants, fixing atmospheric nitrogen and supplying it to the host plant. This is the basis of biological farming and a most important factor of soil fertility (Udvardi & Poole, 2013; Marschner, 2012).
• Agrobacterium: Although Agrobacterium tumefaciens is a pathogen causing crown gall, it has also become a most important tool of plant genetic engineering. Its Ti plasmid is used to transfer genes into plants (Agrios, 2005).
• Azospirillum: Associative nitrogen fixers living in the rhizosphere of cereal crops. They do not form nodules but stimulate plant growth by fixing nitrogen and producing phytohormones (Glick, 2012).

Betaproteobacteria

This class includes several important groups associated with the nitrogen cycle.

• Nitrosomonadales: Ammonia-oxidizing bacteria (AOB), such as Nitrosomonas. They carry out the first stage of nitrification — oxidation of ammonium to nitrite. This is a key process in the soil nitrogen cycle (Prosser, 2008; Scheffer et al., 2018).
• Burkholderiales: This order includes both pathogens (for example, Ralstonia solanacearum — the causative agent of bacterial wilt) and beneficial rhizobacteria that stimulate plant growth (Agrios, 2005; Compant et al., 2005).

Gammaproteobacteria

This class is also incredibly diverse and includes many pathogens and beneficial organisms.

• Pseudomonadales: The genus Pseudomonas is one of the most studied. Among them are both pathogens (for example, Pseudomonas syringae, causing spots and blights), and powerful biocontrol agents, and phosphate-mobilizing bacteria (Agrios, 2005; Lugtenberg & Kamilova, 2009).
• Xanthomonadales: The genus Xanthomonas includes many phytopathogens causing spots, blights, and vascular diseases (for example, black rot of crucifers). It also includes Xylella fastidiosa, causing Pierce's disease of grapevine (Agrios, 2005).
• Enterobacterales: This order includes both pathogens (for example, Erwinia amylovora — the causative agent of fire blight of fruit crops) and beneficial associative nitrogen fixers (for example, Pantoea agglomerans) (Agrios, 2005; Glick, 2012).
• Methanotrophs: Gammaproteobacteria also include aerobic methanotrophs (for example, Methylococcus, Methylomonas), which oxidize methane, reducing its emission from soil. This is an important aspect of greenhouse gas regulation (Conrad, 2007).

Deltaproteobacteria

This class includes organisms with unique metabolic pathways.

• Myxobacteria: Soil predators that form fruiting bodies and hunt other bacteria. They play a role in regulating microbial communities (Weil, 2017).
• Sulfate-reducing bacteria: For example, Desulfovibrio. They reduce sulfate to hydrogen sulfide under anaerobic conditions, participating in the sulfur cycle (Huang et al., 2012).

Epsilonproteobacteria

This group mainly includes human and animal pathogens (for example, Campylobacter, Helicobacter), and its role in the agrosphere is limited. We mention it for completeness.

4.2. Bacillota (Firmicutes) — gram-positive, spore-forming

This phylum, formerly known as Firmicutes, includes gram-positive bacteria with low GC content in DNA. Many of them are capable of forming endospores, which allows them to survive extreme conditions.

1) Bacillus: The genus Bacillus is one of the most important for the agrosphere. It includes:

• PGPB and biocontrol agents: Bacillus subtilis, B. amyloliquefaciens — producers of antibiotics, plant growth stimulators (Lugtenberg & Kamilova, 2009).
• Pathogens: Bacillus cereus can cause food spoilage.
• Entomopathogens: Bacillus thuringiensis (Bt) — a source of toxins used to control insect pests (Weil, 2017).

2) Clostridium: Anaerobic spore-forming bacteria. In soil they participate in decomposition of organic matter and nitrogen fixation (free-living nitrogen fixers). Some species are pathogenic for animals and humans (Weil, 2017; Scheffer et al., 2018).

3) Lactobacillus: Lactic acid bacteria used for ensiling feed and in the food industry. In soil they are less common, but play a role in decomposition of organic matter (Weil, 2017).

4.3. Actinomycetota (Actinobacteria) — gram-positive, mycelial

This phylum, formerly known as Actinobacteria, includes gram-positive bacteria with high GC content. Many of them form mycelium resembling fungal mycelium, but their cells have a prokaryotic structure.

• Streptomyces: The most important genus. These are soil saprophytes that decompose complex organic compounds (cellulose, chitin, lignin). They are also producers of most known antibiotics (streptomycin, tetracycline, etc.). In the agrosphere they play a role in decomposition of organic matter and biocontrol (Weil, 2017; Scheffer et al., 2018).
• Frankia: Actinobacteria that form symbiosis with some woody plants (alder, sea buckthorn), fixing nitrogen. This is an important component of forest and agroforestry ecosystems (Marschner, 2012).
• Mycobacterium: Some species are human and animal pathogens, but in soil they participate in decomposition of organic matter (Weil, 2017).
• Clavibacter: Phytopathogens causing ring rot of potato and bacterial wilt of tomato (Agrios, 2005).

4.4. Bacteroidota (Bacteroidetes) — soil and intestinal

This phylum includes gram-negative bacteria inhabiting various environments.

• Flavobacterium: Soil and aquatic bacteria participating in decomposition of organic matter. Some species can be fish pathogens (Weil, 2017).
• Cytophaga: Soil bacteria decomposing cellulose and chitin. Important for decomposition of plant residues (Weil, 2017).
• Bacteroides: Inhabitants of the intestines of animals and humans, participating in decomposition of complex polysaccharides. They are less common in soil (Weil, 2017).

4.5. Other significant phyla

• Cyanobacteria: Photosynthetic bacteria carrying out oxygenic photosynthesis. In the agrosphere they are important as nitrogen fixers (in symbiosis with Azolla in rice paddies), and also as components of biological crusts (Weil, 2017; Belyakova, 2006).
• Chloroflexi: Green non-sulfur bacteria; some photosynthesize, others are aerobic thermophiles. In soil they participate in decomposition of organic matter (Huang et al., 2012).
• Acidobacteria: A very widespread but poorly studied group of soil bacteria. They dominate in some soils and play an important role in decomposition of organic matter (Weil, 2017).
• Verrucomicrobia: Soil bacteria participating in decomposition of polysaccharides. Their role in the agrosphere is still being studied (Weil, 2017).
• Nitrospirota: Include nitrifying bacteria (for example, Nitrospira), which carry out the second stage of nitrification — oxidation of nitrite to nitrate (Prosser, 2008).

5.1. PGPB (Plant Growth-Promoting Bacteria)

Let us begin with the group of greatest practical interest. PGPB are bacteria that directly or indirectly improve plant growth and development. They can be divided into two large categories: rhizospheric (living in the root zone) and endophytic (living inside plant tissues) (Compant et al., 2005; Glick, 2012).

The mechanisms of PGPB action are diverse:

• Synthesis of phytohormones: For example, Azospirillum produces auxins (indoleacetic acid), which stimulate root growth. This increases the area for water and nutrient uptake (Glick, 2012).
• Reduction of ethylene levels: Some bacteria, for example Pseudomonas putida, contain the enzyme ACC deaminase. This enzyme breaks down the ethylene precursor (ACC), reducing the stress effect of ethylene on the plant. This is especially important under drought, salinity, or pathogen infection (Glick, 2012).
• Improved nutrition: Many PGPB are capable of mobilizing phosphates (converting insoluble phosphates into an available form) or producing siderophores — molecules that bind iron and make it available to the plant (Lugtenberg & Kamilova, 2009).
• Biocontrol: PGPB can suppress phytopathogens through production of antibiotics, competition for nutrients, or induction of systemic resistance (ISR) in plants (Compant et al., 2005; Lugtenberg & Kamilova, 2009).

Typical representatives of PGPB include the genera Azospirillum, Bacillus, Pseudomonas, and Rhizobium.

5.2. Nitrogen fixers — the basis of nitrogen nutrition

Nitrogen is the most important element for life, but atmospheric nitrogen (N₂) is inert and unavailable to most organisms. Nitrogen fixation is the process of reducing N₂ to ammonia (NH₃), which can be carried out only by prokaryotes possessing the enzyme nitrogenase (Marschner, 2012; Udvardi & Poole, 2013).

Nitrogen fixers are divided into several groups:

• Symbiotic nitrogen fixers: These are bacteria that form symbiosis with plants. The most well-known example is rhizobia (Rhizobium, Bradyrhizobium, Sinorhizobium), which form nodules on the roots of leguminous plants. Inside the nodules they reduce N₂ to ammonia, supplying the plant with nitrogen. This is the basis of biological farming (Udvardi & Poole, 2013; Marschner, 2012). Another example is Frankia (Frankia), which forms symbiosis with some woody plants (alder, sea buckthorn) (Marschner, 2012).
• Free-living nitrogen fixers: These bacteria live in soil independently and fix nitrogen without entering into symbiosis. Examples: Azotobacter (aerobe), Clostridium (anaerobe). Their contribution to the soil nitrogen balance is usually smaller than that of symbionts, but they are important in natural ecosystems (Weil, 2017).
• Associative nitrogen fixers: These bacteria live in the rhizosphere or on root surfaces but do not form nodules. Example: Azospirillum. They fix nitrogen using energy from root exudates and can provide up to 30% of a plant's nitrogen requirement (Glick, 2012).

5.3. Methanotrophs — methane consumers

Methanotrophs are aerobic bacteria that use methane (CH₄) as their sole source of carbon and energy. They play a key role in reducing methane emissions — a powerful greenhouse gas — from soil to the atmosphere (Conrad, 2007).

Methanotrophs inhabit the boundary between aerobic and anaerobic zones, where methane formed under anaerobic conditions meets oxygen. In agroecosystems they are especially important in rice paddies and waterlogged soils. Typical representatives: Methylococcus, Methylomonas, Methylosinus (Conrad, 2007; Weil, 2017).

5.4. Methanogens (Archaea) — methane producers

Methanogens are strictly anaerobic archaea that produce methane as the final product of their energy metabolism. They inhabit anaerobic conditions: waterlogged soils, rice paddies, the intestines of ruminants, and sediments of water bodies (Conrad, 2007; Offre et al., 2013).

Methanogens use several substrates:

• Hydrogen and carbon dioxide (hydrogenotrophic methanogens): 4H₂ + CO₂ → CH₄ + 2H₂O.
• Acetate (acetoclastic methanogens): CH₃COOH → CH₄ + CO₂.
• Methylated compounds (methanol, methylamines): methylotrophic methanogens.

In the agrosphere, methanogens play a dual role. On the one hand, they produce methane — a greenhouse gas contributing to climate change. On the other hand, they are part of the natural carbon cycle in anaerobic ecosystems. Typical genera: Methanosarcina, Methanobacterium, Methanococcus (Conrad, 2007; Offre et al., 2013).

5.5. Ammonia-oxidizing archaea (AOA) — key nitrifiers

For a long time it was believed that nitrification — oxidation of ammonium to nitrate — is carried out exclusively by bacteria. However, in 2005 ammonia-oxidizing archaea (AOA) were discovered, which turned out to be much more numerous in soils than their bacterial counterparts (Leininger et al., 2006; Prosser & Nicol, 2008).

AOA belong to the phylum Thaumarchaeota and play a key role in the nitrogen cycle. They carry out the first stage of nitrification: oxidation of ammonium (NH₄⁺) to nitrite (NO₂⁻). Unlike bacteria, AOA are better adapted to low ammonium concentrations and can dominate in oligotrophic soils (Prosser & Nicol, 2008; Leininger et al., 2006).

5.6. Nitrifiers and denitrifiers — the nitrogen cycle

Nitrification is a two-stage process:

1. Ammonia-oxidizing bacteria (AOB) and AOA oxidize ammonium to nitrite. Examples of AOB: Nitrosomonas, Nitrosospira (Prosser, 2008).

2. Nitrite-oxidizing bacteria (NOB) oxidize nitrite to nitrate. Examples: Nitrobacter, Nitrospira (Prosser, 2008).

Denitrification is an anaerobic process in which nitrate (NO₃^-) is reduced to gaseous nitrogen (N₂) through intermediates (NO₂⁻, NO, N₂O). Denitrifiers are facultatively anaerobic bacteria such as Pseudomonas, Bacillus, Paracoccus. This process is important for returning nitrogen to the atmosphere, but it can also lead to nitrogen losses from soil and emission of N₂O — a greenhouse gas (Huang et al., 2012; Scheffer et al., 2018).

5.7. Phosphate- and potassium-mobilizing bacteria

Phosphorus and potassium are essential plant nutrients, but in soil they are often in unavailable form. Phosphate-mobilizing bacteria are capable of converting insoluble phosphates (for example, calcium, iron, aluminum phosphates) into a soluble form available to plants. Mechanisms include production of organic acids (gluconic, citric) that dissolve minerals, or enzymes (phosphatases) that hydrolyze organic phosphates (Glick, 2012; Scheffer et al., 2018).

Potassium-mobilizing bacteria (for example, Bacillus mucilaginosus) are capable of releasing potassium from silicate minerals, making it available to plants (Scheffer et al., 2018).

5.8. Siderophore producers — competition for iron

Iron is necessary for life, but in aerobic soils it is in the Fe³⁺ form, which is extremely insoluble. Siderophores are low-molecular-weight compounds that bind Fe³⁺ with very high affinity and make it available to bacteria. Some PGPB produce siderophores that can suppress phytopathogens by depriving them of iron. This is one of the mechanisms of biocontrol (Lugtenberg & Kamilova, 2009; Glick, 2012).

6.1. Who are archaea? A brief overview of the domain

Let me recall that the domain Archaea was distinguished by Carl Woese in 1977 based on comparison of 16S rRNA sequences (Woese & Fox, 1977). Archaea are prokaryotes, but they fundamentally differ from bacteria:

• Cell wall: Archaea lack peptidoglycan (murein). Their cell walls are built from pseudopeptidoglycan, proteins, or polysaccharides (Weil, 2017).
• Membrane lipids: Archaeal membranes consist of simple ethers with branched hydrocarbon chains (isoprenoids), rather than complex esters with fatty acids, as in bacteria and eukaryotes. This makes them more resistant to extreme conditions (Weil, 2017).
• RNA polymerase: In structure and composition it is closer to the eukaryotic than to the bacterial one (Weil, 2017).
• Metabolism: Archaea possess unique metabolic pathways, including methanogenesis, which is not found in bacteria (Offre et al., 2013).

Modern phylogeny divides the domain Archaea into several main phyla. Traditionally, two large phyla were distinguished: Euryarchaeota and Crenarchaeota. However, with the development of genomics and metagenomics, new phyla were described, such as Thaumarchaeota, Korarchaeota, Aigarchaeota, Geoarchaeota, and others (Offre et al., 2013; Hug et al., 2016). For the agrosphere, the most important are Euryarchaeota (methanogens) and Thaumarchaeota (ammonia-oxidizing archaea).

6.2. Methanogens — methane producers

Methanogens are strictly anaerobic archaea that produce methane (CH₄) as the final product of their energy metabolism. They belong mainly to the phylum Euryarchaeota and include several orders: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, and Methanopyrales (Conrad, 2007; Offre et al., 2013).

Metabolic pathways of methanogenesis:

1. Hydrogenotrophic methanogenesis: Reduction of CO₂ using H₂ as an electron donor:

4H₂ + CO₂ → CH₄ + 2H₂O.

This is the most widespread pathway, characteristic of most methanogens.

2. Acetoclastic methanogenesis: Cleavage of acetate:

CH₃COOH → CH₄ + CO₂.

This pathway is characteristic of representatives of the genera Methanosarcina and Methanosaeta. In rice paddies, acetoclastic methanogenesis accounts for up to two-thirds of total methane production (Conrad, 2007).

3. Methylotrophic methanogenesis: Reduction of methylated compounds (methanol, methylamines) using H₂:

CH₃OH + H₂ → CH₄ + H₂O.

Ecology of methanogens in the agrosphere:

Methanogens inhabit exclusively anaerobic conditions where alternative electron acceptors (O₂, NO₃^-, Fe³⁺, SO₄^2-) are absent. In agroecosystems they occur in:

• Rice paddies: Flooded rice fields are one of the largest anthropogenic sources of methane. Methanogens actively work in the anaerobic soil layer, using organic substances released by rice roots and rice straw (Conrad, 2007; Kimura, 2000).
• Intestines of ruminants: Methanogens are part of the normal microflora of the rumen, where they produce methane, which is released through belching. This is a significant source of methane emission in livestock farming (Conrad, 2007).
• Waterlogged soils and peatlands: In natural and agricultural wetlands, methanogens play a key role in decomposition of organic matter.

Significance for agronomy: Methanogens are of dual interest. On the one hand, they produce methane — a powerful greenhouse gas (its global warming potential is 25 times higher than that of CO₂). On the other hand, they are part of the natural carbon cycle in anaerobic ecosystems. Management of rice fields (for example, periodic drainage) can reduce methane emissions (Conrad, 2007).

6.3. Methanotrophs — methane consumers

Unlike methanogens, methanotrophs are aerobic bacteria that oxidize methane, using it as a source of carbon and energy. We have already mentioned them in the context of ecological groups of bacteria, but here it is important to emphasize their role in the balance of greenhouse gases.

Methanotrophs inhabit the boundary between aerobic and anaerobic zones, where methane formed under anaerobic conditions meets oxygen. In agroecosystems they are especially important in rice paddies, where they consume a significant portion of the methane produced by methanogens, preventing it from reaching the atmosphere (Conrad, 2007; Weil, 2017).

Typical genera of methanotrophs: Methylococcus, Methylomonas, Methylosinus, Methylocystis.

6.4. Ammonia-oxidizing archaea (AOA) — key nitrifiers

This is perhaps the most important group of archaea for the agrosphere after methanogens. Ammonia-oxidizing archaea (AOA) belong to the phylum Thaumarchaeota and carry out the first stage of nitrification — oxidation of ammonium (NH₄⁺) to nitrite (NO₂⁻) (Leininger et al., 2006; Prosser & Nicol, 2008).

History of discovery: For a long time it was believed that nitrification is an exclusively bacterial process. However, in 2005 a group of scientists led by Christof Schleper discovered that in soils archaea, not bacteria, dominate among ammonia-oxidizing prokaryotes (Leininger et al., 2006). This discovery radically changed our ideas about the nitrogen cycle.

Ecology of AOA:

• Distribution: AOA are ubiquitously present in soils, including agricultural soils. They have been found in 12 different soils of three climatic zones, and their abundance exceeded that of bacterial ammonia oxidizers (AOB) by 3000 times in some samples (Leininger et al., 2006).
• Physiology: AOA are better adapted to low ammonium concentrations than AOB. They have higher substrate affinity (low Km values) and can dominate in oligotrophic soils (Prosser & Nicol, 2008).
• Role in soils: AOA play a key role in nitrification, especially in acidic soils and soils with low ammonium content. Their contribution to nitrification can reach 80% in some ecosystems (Prosser & Nicol, 2008; Leininger et al., 2006).

Significance for agronomy: Nitrification is a key process in the nitrogen cycle. It determines which form of nitrogen is available to plants (ammonium or nitrate), and how quickly nitrogen can be lost from soil through leaching or denitrification. Understanding the role of AOA is important for developing strategies for managing nitrogen fertilizers and reducing nitrogen losses (Prosser & Nicol, 2008).

6.5. Halophilic archaea in saline soils

Halophilic archaea are a group of archaea that thrive under conditions of high salinity. They belong to the phylum Euryarchaeota, class Halobacteria. These organisms require high salt concentrations (usually 1.5–5 M NaCl) for growth and are often found in salt lakes, salt ponds, and saline soils (Offre et al., 2013).

Ecology and significance:

• Saline soils: In arid and semiarid regions, where irrigated agriculture is widespread, secondary soil salinization is a serious problem. Halophilic archaea can dominate microbial communities of such soils (Offre et al., 2013).
• Metabolism: Most halophilic archaea are aerobes, heterotrophs. Some of them are capable of photosynthesis using bacteriorhodopsin (halobacteria), but this is not oxygenic photosynthesis.
• Role in the agrosphere: Their role in saline soils is still being studied. They may participate in decomposition of organic matter, but their contribution to soil fertility is limited. However, they are indicators of salinization and can be used to monitor soil condition (Offre et al., 2013).

6.6. Thermophilic and acidophilic archaea in extreme habitats

Although these groups are not typical for the agrosphere, they deserve mention, as they demonstrate the amazing adaptation of archaea to extreme conditions.

• Thermophilic archaea: Inhabit hot springs, hydrothermal vents, volcanic soils. Optimal growth temperatures are 65–110 °C. Examples: Pyrococcus, Thermococcus, Sulfolobus (Offre et al., 2013).
• Acidophilic archaea: Inhabit acidic environments (pH < 3), such as acid mine drainage, volcanic soils. Example: Ferroplasma acidiphilum, Sulfolobus acidocaldarius (Offre et al., 2013).

Significance for the agrosphere: Although these archaea are not typical for agricultural soils, they are important for understanding the evolution of life and can be a source of unique enzymes (for example, thermostable DNA polymerases used in PCR). In addition, acidophilic archaea can participate in the formation of acid mine drainage from rocks, which can be an environmental problem (Offre et al., 2013; Weil, 2017).

6.7. Role of archaea in biogeochemical cycles

To summarize, archaea play a key role in several global biogeochemical cycles:

1. Carbon cycle: Methanogens produce methane, methanotrophs consume it. Archaea also participate in decomposition of organic matter under anaerobic conditions.

2. Nitrogen cycle: AOA carry out the first stage of nitrification, playing a key role in converting ammonium to nitrate.

3. Sulfur cycle: Some archaea (for example, Sulfolobus) oxidize sulfur, others (for example, Archaeoglobus) reduce sulfate.

4. Iron cycle: Some archaea participate in oxidation and reduction of iron.

7.1. Biofertilizers and microbial preparations

One of the most promising directions is the use of biofertilizers (or microbial preparations). These are preparations containing living microorganisms that, when applied to soil or seeds, improve plant nutrition and stimulate their growth (Glick, 2012; Compant et al., 2005).

Main types of biofertilizers:

1. Nitrogen-fixing biofertilizers:

• Rhizobial preparations: Based on nodule bacteria (Rhizobium, Bradyrhizobium). Used for inoculation of seeds of leguminous crops (soybean, pea, alfalfa, clover). This allows reducing or completely abandoning nitrogen fertilizers, since bacteria fix atmospheric nitrogen and supply it to the plant (Udvardi & Poole, 2013; Marschner, 2012). For example, inoculation of soybean can provide up to 200 kg of nitrogen per hectare (Marschner, 2012).
• Associative nitrogen fixers: Based on Azospirillum for cereal crops (wheat, maize, rice). They do not form nodules but live in the rhizosphere and fix nitrogen, and also produce phytohormones (Glick, 2012).
• Free-living nitrogen fixers: Based on Azotobacter for various crops. They are less effective than symbionts but can improve nitrogen nutrition (Weil, 2017).

2. Phosphate-mobilizing biofertilizers:

• Based on bacteria capable of converting insoluble phosphates into an available form. Examples: Bacillus, Pseudomonas. These preparations make it possible to increase the efficiency of phosphorus fertilizers and reduce their doses (Glick, 2012; Scheffer et al., 2018).

3. Complex biofertilizers:

• Contain a mixture of several microorganisms: nitrogen fixers, phosphate mobilizers, potassium mobilizers. This allows comprehensive improvement of plant nutrition (Glick, 2012).

Advantages of biofertilizers:

• Environmentally safe, do not pollute soil and water.
• Reduce costs for mineral fertilizers.
• Improve soil structure and its biological activity.
• Increase plant resistance to stresses (drought, salinity, pathogens) (Glick, 2012; Compant et al., 2005).

Limitations:

• Effectiveness depends on soil and climatic conditions.
• Require proper storage and application.
• Are not always competitive compared with native microflora (Compant et al., 2005).

7.2. Biocontrol of phytopathogens

Biocontrol is the use of living organisms (or their products) to suppress phytopathogens. Bacteria and archaea play a key role here (Lugtenberg & Kamilova, 2009; Compant et al., 2005).

Mechanisms of biocontrol:

1. Antibiosis: PGPB produce antibiotics that kill or suppress the growth of pathogens. Examples: Bacillus subtilis produces subtilin, Pseudomonas fluorescens — phenazine (Lugtenberg & Kamilova, 2009).

2. Competition for nutrients and niches: PGPB quickly colonize the root system, occupying places that could be occupied by pathogens. They also compete for nutrients, depriving pathogens of resources (Lugtenberg & Kamilova, 2009).

3. Parasitism and lysis: Some bacteria (for example, Streptomyces) produce enzymes (chitinases, glucanases) that destroy the cell walls of fungal pathogens (Compant et al., 2005).

4. Induction of systemic resistance (ISR): PGPB can "switch on" the plant's defense mechanisms, making it more resistant to pathogens. This occurs without direct contact between the bacterium and the pathogen (Compant et al., 2005; Lugtenberg & Kamilova, 2009).

5. Siderophores: PGPB produce siderophores that bind iron, making it unavailable to pathogens (Lugtenberg & Kamilova, 2009).

Examples of biocontrol preparations:

• Based on Bacillus subtilis — for protection against root rots, powdery mildew.
• Based on Pseudomonas fluorescens — for protection against bacterial and fungal pathogens.
• Based on Trichoderma (fungus) — for protection against a wide range of pathogens (Lugtenberg & Kamilova, 2009).

7.3. Soil health indicators

Microorganisms can serve as bioindicators of soil condition. Their abundance, diversity, and activity reflect soil health and its ability to perform its functions (Weil, 2017; Scheffer et al., 2018).

Main microbial indicators:

1. Microbial biomass: The total mass of microorganisms in the soil. High biomass usually indicates healthy, fertile soil (Weil, 2017).

2. Microbial respiration: Release of CO₂ by the soil. Reflects the overall metabolic activity of the microbial community (Weil, 2017).

3. Enzymatic activity: Activity of soil enzymes (dehydrogenases, phosphatases, ureases) — an indicator of the intensity of biochemical processes (Huang et al., 2012).

4. Diversity of the microbial community: Assessed using molecular methods (16S rRNA sequencing). High diversity is a sign of sustainable soil (Weil, 2017).

5. Fungus-to-bacterium ratio: Different ratios are characteristic of different soil types and farming systems. For example, no-till systems promote an increase in the proportion of fungi (Weil, 2017).

6. Presence of specific groups: For example, the presence of AOA (ammonia-oxidizing archaea) may indicate active nitrification (Prosser & Nicol, 2008).

7.4. Bioremediation of pollutants

Microorganisms are capable of degrading or detoxifying environmental pollutants. This is used in bioremediation — the technology of cleaning soils and waters with the help of biological agents (Weil, 2017; Glick, 2012).

Main directions:

1. Degradation of organic pollutants: Some bacteria (for example, Pseudomonas, Bacillus, Rhodococcus) are capable of degrading petroleum products, pesticides, and polycyclic aromatic hydrocarbons (PAHs) (Weil, 2017; Huang et al., 2012).

2. Detoxification of heavy metals: Bacteria can immobilize or transform heavy metals, reducing their toxicity. For example, sulfate-reducing bacteria precipitate metals in the form of sulfides (Huang et al., 2012).

3. Phytoremediation using PGPB: Combined use of plants and PGPB for soil cleaning. Bacteria stimulate the growth of plants that accumulate pollutants and help them cope with stress (Glick, 2012).

4. Bioremediation using archaea: Methanogens and other archaea can participate in the degradation of organic pollutants under anaerobic conditions (Offre et al., 2013).

7.5. Impact of agrotechnologies on microbial communities

Understanding how agrotechnologies affect microbial communities makes it possible to manage them to increase fertility and sustainability of agroecosystems (Weil, 2017; Scheffer et al., 2018).

Main factors of influence:

1. Tillage:

• Conventional (plowing): Destroys microbial communities, reduces diversity, promotes loss of organic carbon (Weil, 2017).
• No-till: Preserves soil structure, increases microbial biomass and diversity, promotes accumulation of organic carbon (Weil, 2017).

2. Fertilizer application:

• Mineral fertilizers: Can stimulate the growth of certain groups of microorganisms, but in excess — reduce diversity and cause soil acidification (Weil, 2017; Scheffer et al., 2018).
• Organic fertilizers: Increase microbial biomass and activity, improve soil structure (Weil, 2017).

3. Pesticides:

• Can have a toxic effect on non-target microorganisms, reduce diversity and activity (Weil, 2017).
• Some pesticides can be degraded by microorganisms, but at the same time can accumulate in food chains (Weil, 2017).

4. Crop rotation:

• Diverse crop rotations contribute to increased microbial diversity and soil sustainability (Weil, 2017).
• Monoculture reduces diversity and can lead to accumulation of pathogens (Weil, 2017).

5. Irrigation:

• Affects soil moisture, which determines the activity of microorganisms. Waterlogging can lead to anaerobic conditions and the development of methanogens (Conrad, 2007).

8.1. Prokaryotes — the foundation of agroecosystems

The main conclusion we must draw is that prokaryotes are the foundation of agroecosystems. Their role cannot be overestimated. Let us recall key figures and facts:

• One gram of soil contains from several billion to hundreds of billions of bacterial cells (Weil, 2017).
• The biomass of microorganisms in the upper soil layer can reach several tons per hectare, which is comparable to the biomass of a large herd of animals (Weil, 2017).
• Prokaryotes dominate in the processes of decomposition of organic matter, the cycles of nitrogen, phosphorus, and sulfur (Huang et al., 2012; Scheffer et al., 2018).
• Without their activity, soil fertility would be impossible, since the main nutrients would remain "locked" in dead organic matter.

It is prokaryotes that provide those ecosystem services on which agriculture depends: mineralization of organic matter, nitrogen fixation, phosphorus mobilization, biocontrol of pathogens, and much more.

8.2. Taxonomic diversity and modern systematics

We have seen that the world of prokaryotes is enormous and diverse. It is divided into two fundamental domains — Bacteria and Archaea, which diverged in evolution billions of years ago (Woese & Fox, 1977). We became acquainted with the main phyla significant for the agrosphere:

• Pseudomonadota (Proteobacteria) — the most diverse phylum, including rhizobia, pseudomonads, xanthomonads, and enterobacteria (Agrios, 2005; Udvardi & Poole, 2013).
• Bacillota (Firmicutes) — gram-positive spore-forming bacteria, including Bacillus and Clostridium (Weil, 2017).
• Actinomycetota (Actinobacteria) — mycelial bacteria, including Streptomyces and Frankia (Weil, 2017; Scheffer et al., 2018).
• Bacteroidota — soil and intestinal bacteria participating in the decomposition of organic matter (Weil, 2017).
• Cyanobacteria, Acidobacteria, Verrucomicrobia, and others (Huang et al., 2012; Weil, 2017).

We also discussed that modern systematics of prokaryotes is based on molecular data. The 16S rRNA gene was for a long time the "gold standard," but today it is being replaced by whole-genome methods (ANI, concatenated genes), which make it possible to construct more accurate phylogenetic trees and determine species boundaries (Doolittle & Papke, 2006; Yoon et al., 2017; Parks et al., 2018). Databases SILVA, EzBioCloud, and GTDB have become indispensable tools for the identification and classification of microorganisms (Quast et al., 2012; Yoon et al., 2017; Parks et al., 2018).

8.3. Ecological groups and their role in the agrosphere

We examined the main ecological groups of prokaryotes that determine the functioning of agroecosystems:

• PGPB (Plant Growth-Promoting Bacteria): Azospirillum, Bacillus, Pseudomonas, Rhizobium — stimulate plant growth through the synthesis of phytohormones, improvement of nutrition, and biocontrol (Glick, 2012; Compant et al., 2005; Lugtenberg & Kamilova, 2009).
• Nitrogen fixers: symbiotic (rhizobia, frankiae), free-living (Azotobacter, Clostridium), associative (Azospirillum) — provide nitrogen nutrition for plants (Marschner, 2012; Udvardi & Poole, 2013).
• Methanotrophs: consume methane, reducing greenhouse gas emissions (Conrad, 2007).
• Methanogens (Archaea): produce methane under anaerobic conditions — rice fields, intestines of ruminants (Conrad, 2007; Offre et al., 2013).
• Ammonia-oxidizing archaea (AOA): key nitrifiers dominating in soils (Leininger et al., 2006; Prosser & Nicol, 2008).
• Nitrifying and denitrifying bacteria: provide the nitrogen cycle (Prosser, 2008; Huang et al., 2012).
• Phosphate-mobilizing and potassium-mobilizing bacteria: convert elements into a form available to plants (Glick, 2012; Scheffer et al., 2018).
• Siderophore producers: compete for iron, suppressing pathogens (Lugtenberg & Kamilova, 2009).

We also dwelt in detail on archaea, which, as it turned out, are not an exotic group but are ubiquitously present in soils and play a key role in biogeochemical cycles (Offre et al., 2013; Leininger et al., 2006).

8.4. Practical significance for agronomy

We discussed how knowledge about prokaryotes is applied in practice:

• Biofertilizers: preparations based on nitrogen fixers, phosphate mobilizers, and PGPB make it possible to reduce dependence on mineral fertilizers and increase yield (Glick, 2012).
• Biocontrol: the use of microorganisms to suppress phytopathogens through antibiosis, competition, parasitism, and induction of systemic resistance (Lugtenberg & Kamilova, 2009; Compant et al., 2005).
• Soil health indicators: microbial biomass, respiration, enzymatic activity, and diversity are used to assess soil condition (Weil, 2017; Huang et al., 2012).
• Bioremediation: microorganisms degrade pollutants (petroleum products, pesticides) and detoxify heavy metals (Weil, 2017; Glick, 2012).
• Impact of agrotechnologies: tillage, fertilizers, pesticides, and crop rotation have a significant impact on microbial communities, which must be taken into account when managing agroecosystems (Weil, 2017; Scheffer et al., 2018).

8.5. Limitations and prospects

Despite enormous progress, our knowledge about prokaryotes is far from complete. The majority of microorganisms (more than 99%) cannot be cultivated under laboratory conditions, and we know about them only from molecular data (Weil, 2017). This creates serious limitations for studying their physiology and ecology.

In addition, horizontal gene transfer and genome mosaicism make the construction of a single evolutionary tree a complex task (Doolittle & Papke, 2006). Methods of metagenomics, metatranscriptomics, and metabolomics open new possibilities for studying uncultivated microorganisms and their functions under natural conditions (Hug et al., 2016).

Prospects for further research include:

• Development of new cultivation methods for access to "microbial dark matter."
• Use of stable isotopes (SIP) to identify active microorganisms in soil (Huang et al., 2012).
• Application of synthetic biology to create effective microbial consortia for agronomy.
• Study of the interaction of bacteria and archaea with plants and other microorganisms within the framework of the holobiont (Huang et al., 2012).

8.6. Concluding remarks

Dear students, we have completed the first module of our course. You have received fundamental knowledge about the most ancient, numerous, and metabolically diverse group of organisms on Earth — prokaryotes. You have learned that bacteria and archaea are not simply "microbes" but complex, highly organized systems that determine life on our planet.

For the agronomist, this knowledge has direct practical significance. Understanding soil microbiology makes it possible to:

• Effectively manage fertility, reducing fertilizer costs.
• Protect plants from diseases using biological methods.
• Assess soil health and predict its productivity.
• Develop sustainable farming systems that conserve resources for future generations.

Modern systematics and taxonomy are not simply the "science of names." They are a tool that allows us to understand evolutionary relationships, predict the properties of organisms, and use them effectively for practical purposes. Databases such as SILVA, EzBioCloud, and GTDB are not reference books but working tools that will accompany you throughout your professional activity.