Eukaryotic microorganisms (protists) of agroecosystems

Amoebozoa

The supergroup Amoebozoa includes amoebae and slime molds. These organisms are characterized by the ability to form pseudopodia — temporary outgrowths of cytoplasm used for movement and food capture. Among Amoebozoa there are both free-living forms and parasites (Adl et al., 2012; Ruppert et al., 2008).

In soil ecosystems, amoebae are among the most numerous and diverse protists. They actively participate in regulating bacterial abundance and in nutrient cycling (Geisen et al., 2018).

Excavata

The supergroup Excavata unites flagellates with a characteristic cell structure that includes the so-called “excavate” groove — a depression on the cell surface used for feeding. This group includes both free-living forms and parasites, including causative agents of serious human and animal diseases (for example, Giardia, Trichomonas) (Adl et al., 2012; Hickman et al., 2004).

In soils, Excavata are represented mainly by heterotrophic flagellates that feed on bacteria and participate in the decomposition of organic matter (Geisen et al., 2018).

SAR (Stramenopiles, Alveolata, Rhizaria)

The supergroup SAR is one of the largest and most diverse groups of eukaryotes. It includes three main lineages (Adl et al., 2012; Geisen et al., 2018):

Stramenopiles (or Heterokonta) — a group characterized by the presence of two different flagella (one with tripartite tubular hairs, the other smooth). Stramenopiles include diatoms, brown algae, oomycetes (including Phytophthora infestans — the causal agent of potato late blight), and other groups.

Alveolata — a group uniting ciliates (Ciliophora), apicomplexans (Apicomplexa, including malaria pathogens — Plasmodium), and dinoflagellates (Dinoflagellata). A characteristic feature of Alveolata is the presence of alveoli (membrane sacs) beneath the cell membrane (Adl et al., 2012; Hickman et al., 2004).

Rhizaria — a group including foraminiferans, radiolarians, cercozoans, and phytomyxids (including Plasmodiophora brassicae — the causal agent of clubroot of cabbage). Many Rhizaria form thin pseudopodia (filopodia or reticulopodia) for feeding and movement (Adl et al., 2012; Geisen et al., 2018).

Archaeplastida

The supergroup Archaeplastida includes organisms whose plastids originated through primary endosymbiosis with a cyanobacterium. This group includes red algae, green algae, and higher plants. In the context of protists, we are primarily interested in unicellular green algae, which occur in soils and water bodies (Adl et al., 2012; Raven et al., 2013).

Other supergroups

In addition to those listed, supergroups Cryptista and Haptista are also distinguished, as well as a number of organisms of unclear systematic position. Some of them occur in soils, but their role in agroecosystems has been insufficiently studied (Adl et al., 2012).

Naked amoebae

Naked amoebae are organisms lacking a shell or theca. Their cell is surrounded only by a plasma membrane, which allows them to change body shape and actively move through soil pores (Bonkowski et al., 2012).

In soils, naked amoebae are represented by several morphological types. The largest of them, such as Amoeba proteus, reach 250–600 µm in diameter and are well known as classical objects for studying amoeboid movement (Hickman et al., 2004). However, smaller forms predominate in soils, with sizes of 15–100 µm in width and a thickness of only about 1 µm when attached to a surface (Bonkowski et al., 2012).

Among naked amoebae, several ecological groups are distinguished. Some species, for example Acanthamoeba and Naegleria, are facultative pathogens of humans and animals, which is of sanitary importance (Geisen et al., 2018; Bonkowski et al., 2012). Others, such as Hartmannella, inhabit exclusively soil and water, feeding on bacteria.

It is important to note that naked amoebae can serve as a reservoir for pathogenic bacteria. As Geisen et al. (2018) note, bacteria such as Legionella, Chlamydia, Listeria, and Salmonella can survive inside amoeba cells, which protects them from unfavorable environmental conditions and disinfectants. This phenomenon has been called the “Trojan horse” of the microbial world (Bonkowski et al., 2012).

Testate amoebae

Testate amoebae are distinguished by the presence of a shell (test), which can be built from organic material, silica, or mineral particles glued together by organic cement (Bonkowski et al., 2012; Ruppert et al., 2008).

Testate amoebae are widespread in soils, especially in moist and acidic habitats such as bogs, peatlands, and forest litter. Their abundance can reach 30,000 individuals per gram of dry soil (Bonkowski et al., 2012). In agroecosystems, testate amoebae are less common than naked amoebae, but they still play an important role in soil processes.

Among testate amoebae, several main genera are distinguished. Arcella has a dome-shaped shell made of organic material (Ruppert et al., 2008). Difflugia builds a shell from mineral particles captured from the environment (Ruppert et al., 2008; Hickman et al., 2004). Euglypha has a shell made of siliceous scales (Ruppert et al., 2008). Trinema and Corythion are small forms with a characteristic shell (Bonkowski et al., 2012).

Testate amoebae play an important role in the silicon cycle in soils. As Geisen et al. (2018) note, hyalosphenid testate amoebae that feed on euglyphids participate in the silicon cycle, and their evolutionary radiations coincide with major changes in terrestrial vegetation and climate.

Slime molds (myxomycetes)

Slime molds, or myxomycetes (Myxomycetes), are a unique group of protists that at different stages of the life cycle are represented by both unicellular and multinucleate forms (Ruppert et al., 2008; Hickman et al., 2004).

The plasmodial stage of myxomycetes is a multinucleate mass of cytoplasm lacking a cell wall, capable of amoeboid movement and phagocytosis. The plasmodium can reach several centimeters in diameter and is clearly visible to the naked eye (Raven et al., 2013). In soils, myxomycetes occur in forest litter, on decaying wood, and in the upper soil horizons (Geisen et al., 2018).

Under unfavorable conditions, the plasmodium transforms into fruiting bodies (sporangia) containing spores. Spores can persist in soil for a long time and germinate when favorable conditions occur (Ruppert et al., 2008).

In agroecosystems, myxomycetes are less common than in natural habitats, since they are sensitive to mechanical tillage and fertilizer application. However, they may play a certain role in the decomposition of organic matter and regulation of bacterial abundance (Geisen et al., 2018).

It should be noted that myxomycetes are not plant pathogens, although some species can damage cultivated plants during mass reproduction on the soil surface (Ruppert et al., 2008).

Heterotrophic flagellates

Heterotrophic flagellates are one of the most numerous groups of soil protists. Their abundance in soils can reach 10⁴–10⁵ individuals per gram (Bonkowski et al., 2012).

Most soil flagellates are small — 4–15 µm in length (Bonkowski et al., 2012). They are able to squeeze through pores only 3 µm in diameter thanks to their flexible cell membrane. Flagella are used not only for swimming but also for creating a feeding current that attracts bacterial cells.

Among heterotrophic flagellates, several ecological groups are distinguished. Cercomonads are among the most abundant soil flagellates, capable of both swimming and attaching to surfaces (Geisen et al., 2018). Glissomonads are the dominant gliding flagellates in soils (Geisen et al., 2018). Kinetoplastids include both free-living forms (for example, Bodo) and parasites (for example, Trypanosoma, Leishmania) (Ruppert et al., 2008).

Heterotrophic flagellates play a key role in the microbial loop, controlling bacterial abundance and releasing nutrients in forms available to plants (Bonkowski et al., 2012; Clarholm, 1985).

Phototrophic flagellates

Phototrophic flagellates, or unicellular algae, are capable of photosynthesis due to the presence of chloroplasts. In soils, they occur mainly in the upper horizons where light penetrates (Raven et al., 2013; Geisen et al., 2018).

Among soil phototrophic flagellates, green algae (Chlorophyta) are the most common, such as Chlamydomonas, Chlorella, and Scenedesmus (Raven et al., 2013). They can form symbiotic associations with fungi (lichens) or with other organisms (Geisen et al., 2018).

Diatoms (Bacillariophyceae) also occur in soils, especially in moist habitats. Their siliceous frustules are well preserved in soil and can be used for paleoecological reconstructions (Geisen et al., 2018).

Phototrophic protists contribute to the primary production of soil ecosystems, especially in arid and semiarid regions, where they form biological crusts (biocrusts) (Geisen et al., 2018; Weil, 2017).

Predatory ciliates

Predatory ciliates feed on other protists, including ciliates, flagellates, and amoebae. They play an important role in regulating the abundance of other microorganisms (Ruppert et al., 2008).

A classic example of a predatory ciliate is Didinium, which feeds exclusively on paramecia. Didinium has a barrel-shaped body with a characteristic proboscis used to capture prey. Upon contact with a paramecium, Didinium fires toxicysts that paralyze the victim, and then swallows it whole (Ruppert et al., 2008; Hickman et al., 2004).

Other predatory ciliates, such as Dileptus and Litonotus, also use toxicysts to immobilize prey (Ruppert et al., 2008). In soils, predatory ciliates can regulate the abundance of bacterivorous and detritivorous forms, influencing energy flows in the soil food web (Geisen et al., 2018).

Bacterivorous ciliates

Bacterivorous ciliates feed on bacteria, creating water currents with the help of cilia that direct bacterial cells into the cell mouth (cytostome) (Ruppert et al., 2008; Hickman et al., 2004).

A typical representative of bacterivorous ciliates is Paramecium, which feeds on bacteria, algae, and other small organisms (Hickman et al., 2004). In soils, bacterivorous ciliates are represented mainly by small forms, such as Colpoda, Cyclidium, and Uronema (Bonkowski et al., 2012; Geisen et al., 2018).

Bacterivorous ciliates play a key role in the microbial loop, releasing nitrogen and phosphorus in forms available to plants (Bonkowski et al., 2012; Clarholm, 1985). As Bonkowski et al. (2012) note, ciliates have a short generation time (several hours with sufficient food) and a high rate of bacterial consumption, which makes them an important link in the soil food web.

Detritivores

Detritivorous ciliates feed on organic particles and decomposing plant and animal remains. They play an important role in the decomposition of organic matter, accelerating its mineralization (Bonkowski et al., 2012).

Detritivores are usually larger than bacterivorous ciliates and have a more complexly organized oral apparatus (Ruppert et al., 2008). They can feed both on organic particles directly and on microorganisms developing on them.

In soils, detritivores are represented by genera such as Stentor, Spirostomum, and Blepharisma (Ruppert et al., 2008). They can reach considerable sizes (up to 2 mm) and make a substantial contribution to organic matter decomposition.

Testate amoebae (euglyphids)

Euglyphids (Euglyphida) are testate amoebae belonging to cercozoans. Their shell is built from siliceous scales, which can be round, oval, or elongated (Ruppert et al., 2008; Geisen et al., 2018).

Euglyphids are widespread in soils, especially in moist and acidic habitats. They feed on bacteria, fungi, and small protists, using thin filopodia that emerge through the shell aperture (Ruppert et al., 2008; Geisen et al., 2018).

Among euglyphids, the most common genera are Euglypha, Trinema, Assulina, and Placocista (Ruppert et al., 2008; Bonkowski et al., 2012). They play an important role in the silicon cycle, since their shells are built from silicon, and after cell death the silicon returns to the soil (Geisen et al., 2018).

Heliozoans

Heliozoans (Heliozoa) are a group of protists characterized by a spherical body shape and numerous thin pseudopodia (axopodia) radiating from the cell (Ruppert et al., 2008; Hickman et al., 2004).

The axopodia of heliozoans are supported by bundles of microtubules and are used for capturing prey as well as for floating in water (Ruppert et al., 2008). Some heliozoans have a stalk for attachment to a substrate (Ruppert et al., 2008).

In soils, heliozoans are less common than in water bodies, but they can still be found in moist habitats. They feed on bacteria, other protists, and small invertebrates (Ruppert et al., 2008; Geisen et al., 2018).

Among soil heliozoans, the most common genera are Actinophrys, Actinosphaerium, and Clathrulina (Ruppert et al., 2008). They play the role of predators in the soil food web, regulating the abundance of other microorganisms.

Diatoms

Diatoms (Bacillariophyceae) are a group of photosynthetic protists characterized by a siliceous frustule consisting of two valves (Raven et al., 2013).

In soils, diatoms occur in the upper horizons where there is enough light for photosynthesis. They can form biological crusts on the soil surface, especially in arid regions (Geisen et al., 2018).

Diatoms play an important role in the silicon cycle in soils. Their siliceous frustules persist in soil for a long time and can be used for paleoecological studies (Geisen et al., 2018).

Green algae

Green algae (Chlorophyta) are the most diverse group of soil algae. They contain chlorophylls a and b and store starch (Raven et al., 2013).

In soils, green algae are represented by unicellular and colonial forms. The most common genera are Chlamydomonas, Chlorella, Scenedesmus, and Stichococcus (Raven et al., 2013; Geisen et al., 2018).

Green algae can enter into symbiotic relationships with fungi, forming lichens. Lichens play an important role in soil formation, especially on primary substrates (Raven et al., 2013).

Cyanobacteria

Cyanobacteria are prokaryotic photosynthetic organisms that were traditionally considered together with algae (Raven et al., 2013; Weil, 2017).

Cyanobacteria are widespread in soils, especially in rice paddies and other waterlogged habitats. They are capable of fixing atmospheric nitrogen, which makes them an important component of the nitrogen cycle in agroecosystems (Weil, 2017).

Some cyanobacteria form symbiotic associations with plants (for example, with the aquatic fern Azolla) and are used as biofertilizers (Weil, 2017).

Microscopy

Traditional methods for studying protists are based on light microscopy. For identification, morphological characters are used: cell shape, size, presence and structure of the shell, arrangement of flagella or cilia, and structure of the nuclear apparatus (Bonkowski et al., 2012; Ruppert et al., 2008).

To study the fine structure of the cell, electron microscopy (transmission and scanning) is used, which reveals ultrastructural characters important for taxonomy (Ruppert et al., 2008; Geisen et al., 2018).

However, microscopy has limitations: many protists are morphologically indistinguishable (cryptic species), and their identification requires high researcher qualification (Geisen et al., 2018; Lentendu et al., 2022).

Cultivation

Cultivating protists on artificial media allows obtaining pure cultures for detailed study. To isolate protists from soil, dilution, enrichment, and cloning methods are used (Bonkowski et al., 2012; Geisen et al., 2018).

However, most soil protists cannot be cultivated on standard media, which limits the application of this method (Geisen et al., 2018).

Molecular methods

Modern molecular methods, primarily analysis of 18S rRNA gene sequences, make it possible to reveal protist diversity without culturing them (Lentendu et al., 2022; Geisen et al., 2018).

Metabarcoding is a method based on amplification and sequencing of a specific region of the 18S rRNA gene (for example, the V4 or V9 regions) from total DNA extracted from soil (Lentendu et al., 2022). This approach makes it possible to detect hundreds and thousands of operational taxonomic units (OTUs) in a single soil sample.

For sequence identification, specialized databases are used, such as PR² (Protist Ribosomal Reference database) and SILVA (Quast et al., 2012; Lentendu et al., 2022). These databases contain curated 18S rRNA sequences with taxonomic information.

Metabarcoding has a number of limitations: uneven amplification of different groups, the presence of introns in the 18S rRNA gene, and incompleteness of databases (Lentendu et al., 2022; Geisen et al., 2018). Nevertheless, it is the most powerful method for studying the diversity of soil protists.

Problems of protist identification

Identification of soil protists involves a number of problems (Geisen et al., 2018; Lentendu et al., 2022):

Cryptic species. Many protists are morphologically indistinguishable but genetically and ecologically distinct. This complicates assessment of true diversity.

Polymorphism. Some protists have complex life cycles with several morphologically different stages, which complicates their identification.

Lack of universal markers. There is no single molecular marker suitable for identifying all groups of protists.

Incompleteness of databases. Many sequences obtained from the environment have no close matches in databases, which complicates their taxonomic identification.

The concept of the microbial loop

The key concept describing the role of protists in soil processes is the “microbial loop.” This concept was originally developed for aquatic ecosystems but was later adapted for soils through the work of the Swedish researcher Marianne Clarholm (Clarholm, 1985).

The essence of the microbial loop in soil is as follows. Plants release organic compounds through their roots (root exudates), which stimulate bacterial growth in the rhizosphere. Bacteria, in turn, immobilize (retain) nitrogen and other nutrients in their biomass. If bacteria were not consumed by other organisms, these nutrients would remain “locked” in bacterial cells and would be unavailable to plants (Clarholm, 1985; Bonkowski et al., 2012).

Protists, by feeding on bacteria, “open” this loop: they consume bacterial biomass, and the excess nutrients (primarily nitrogen) they cannot use for their own growth are released into the environment in a form available to plants. Thus, protists act as a key link connecting bacterial production with plant needs (Clarholm, 1985; Bonkowski et al., 2012; Geisen et al., 2018).

Scale of bacterial consumption by protists

The scale of bacterial consumption by protists in soils is enormous. As Bonkowski et al. (2012) note, tens of thousands of protist individuals can inhabit one gram of soil, each consuming hundreds and thousands of bacterial cells per hour.

For illustration: one Paramecium ciliate can consume up to 1,000 bacterial cells per hour, while amoebae and flagellates consume hundreds of cells per hour (Bonkowski et al., 2012). Considering that protist abundance in soil can reach 10⁴–10⁵ individuals per gram, total bacterial consumption constitutes a significant share of bacterial production (Geisen et al., 2018).

Studies show that protists can consume from 20 to 50% of bacterial production in soils (Bonkowski et al., 2012). This means that without protists, bacterial abundance would be significantly higher, and their community would differ in composition.

Selective feeding and structure of bacterial communities

It is important to emphasize that protists do not feed on all bacteria indiscriminately. As Geisen et al. (2018) note, protists display selectivity in feeding, preferring certain groups of bacteria.

Studies have shown that protists prefer bacteria without pigments, toxins, filaments, and external mucous coats (Bonkowski et al., 2012). Gram-negative bacteria, as a rule, are preferred over gram-positive ones (Bonkowski et al., 2012). In addition, the size of bacterial cells matters: larger cells are consumed more actively than smaller ones (Geisen et al., 2018).

Selective feeding by protists leads to the formation of a certain structure of bacterial communities. As experiments have shown, the presence of different species of amoebae and flagellates leads to the formation of different bacterial communities (Geisen et al., 2018). This occurs because some bacteria are actively consumed, while others avoid predation and gain a competitive advantage (Geisen et al., 2018; Bonkowski et al., 2012).

Moreover, bacteria have evolved diverse defensive strategies against protists: changes in cell size, colony formation, release of toxic compounds, and active movement (Geisen et al., 2018; Fujino et al., 2025). These defense mechanisms, in turn, can influence the composition of bacterial communities and their functional properties.

Interaction with bacterial antagonists of phytopathogens

Of particular interest is the interaction of protists with bacteria that suppress phytopathogens. As studies by Jousset et al. (2006, 2010) showed, the presence of protists stimulates the production by bacteria of secondary metabolites, including antibiotics and toxins that suppress pathogen growth.

This phenomenon has been called the “indirect mechanism” of pathogen suppression (Fujino et al., 2025). Its essence is that protists, by consuming bacteria, create selective pressure that favors bacteria producing protective compounds. These compounds, in turn, suppress the growth of phytopathogenic fungi and oomycetes (Fujino et al., 2025; Geisen et al., 2018).

Thus, protists not only regulate bacterial abundance but also influence their functional properties, stimulating the development of bacterial antagonists of phytopathogens.

Mechanisms of nutrient release

One of the most important functions of protists in soil is their participation in nutrient cycling. As already noted, protists consume bacteria and release the excess nutrients they cannot use for their own growth into the environment (Clarholm, 1985; Bonkowski et al., 2012).

This process is due to the fact that the C:N ratio of protists is higher than that of their bacterial food. Bacteria have a C:N ratio of about 3:1–4:1, whereas protists have about 8:1–12:1 (Bonkowski et al., 2012). This means that when consuming bacterial biomass, protists obtain an excess of nitrogen relative to carbon, and this excess is released in the form of ammonium (NH₄⁺) (Clarholm, 1985; Bonkowski et al., 2012).

Phosphorus and other nutrients are released in a similar way. As Bonkowski et al. (2012) note, protists release phosphorus in a form available to plants, which is especially important in soils with low available phosphorus.

Quantitative estimates

Quantitative estimates of the contribution of protists to nutrient cycling are impressive. As Clarholm (1985) notes, in some ecosystems protists can release up to 30–40% of the nitrogen available to plants.

Studies in the rhizosphere have shown that the presence of protists increases nitrogen mineralization by 20–50% (Bonkowski et al., 2012). This occurs both through direct ammonium release by protists and through stimulation of bacterial activity (Clarholm, 1985; Bonkowski et al., 2012).

It is important to emphasize that nutrient release by protists occurs in close proximity to plant roots, since protists concentrate in the rhizosphere due to the abundance of bacterial food (Bonkowski et al., 2012). This makes nutrients immediately available to plants.

Interaction with mycorrhizal fungi

Of particular interest is the interaction of protists with mycorrhizal fungi — plant symbionts that improve plant supply of phosphorus and other nutrients.

As Geisen et al. (2018) note, arbuscular mycorrhizal (AM) fungi do not possess enzymatic systems for mineralizing organic nitrogen and depend on other microorganisms for the supply of mineral nitrogen. Protists, by consuming bacteria, release ammonium, which is then absorbed by mycorrhizal hyphae and transferred to the plant (Geisen et al., 2018).

Thus, protists and mycorrhizal fungi act synergistically, providing the plant with nitrogen and phosphorus (Geisen et al., 2018; Bonkowski et al., 2012).

Effect on carbon cycling

In addition to nitrogen and phosphorus, protists influence carbon cycling in soil. By consuming bacteria, they release CO₂ during respiration, which increases the total CO₂ flux from soil to the atmosphere (Bonkowski et al., 2012).

In addition, protists promote carbon sequestration in soil. As Geisen et al. (2018) note, mycophagous protists feeding on fungi can promote stabilization of organic matter, since fungal mycelium not consumed by protists can become incorporated into soil aggregates and persist for a long time.

Direct and indirect mechanisms of plant growth stimulation

Protists exert a significant stimulatory influence on plant growth. This influence can be both direct (through the release of phytohormones) and indirect (through improving plant nutrition and soil structure) (Bonkowski et al., 2012; Geisen et al., 2018).

As Bonkowski et al. (2012) note, even early studies showed that the presence of protists increases plant growth by 20–50% compared with a control where protists were absent. This effect was observed in different plant species, including cereals, legumes, and vegetable crops.

Release of phytohormones and signaling molecules

One mechanism of plant growth stimulation is the release of phytohormones and signaling molecules by protists. As studies by Krome et al. (2010) showed, the presence of the amoeba Acanthamoeba castellanii led to an increase in the content of free auxins in plants, which stimulated lateral root growth.

Auxins are a class of phytohormones that regulate plant growth and development. An increase in auxin content under the influence of protists leads to the formation of a more branched root system, which improves water and nutrient uptake (Bonkowski et al., 2012; Geisen et al., 2018).

In addition, protists can influence the synthesis of cytokinins and other phytohormones, although the mechanisms of this influence have been insufficiently studied (Bonkowski et al., 2012).

Improvement of soil structure

Protists also contribute to improving soil structure. As Geisen et al. (2018) note, protists, by consuming bacteria, release mucus and other organic compounds that glue soil particles into aggregates.

In addition, protists, moving through soil pores, promote mixing of organic and mineral material, which improves soil structure (Bonkowski et al., 2012).

Testate amoebae and ciliates play a special role in improving soil structure, as they release significant amounts of organic compounds (Bonkowski et al., 2012; Geisen et al., 2018).

Effect on nutrient availability

As already noted, protists increase the availability of nitrogen and phosphorus to plants. This occurs both through direct release of nutrients and through stimulation of the activity of bacteria and fungi involved in organic matter mineralization (Clarholm, 1985; Bonkowski et al., 2012).

Studies have shown that plants growing in the presence of protists contain more nitrogen and phosphorus in their tissues than plants growing without protists (Bonkowski et al., 2012; Geisen et al., 2018). This indicates that protists improve plant nutrition.

Direct mechanism of pathogen suppression

One of the most promising functions of protists in agroecosystems is their ability to suppress phytopathogens. As Fujino et al. (2025) note, protists can suppress pathogens by two mechanisms: direct and indirect.

The direct mechanism consists in the fact that predatory protists consume phytopathogenic microorganisms — fungi, oomycetes, and bacteria. As studies have shown, many protists are capable of feeding on spores and mycelium of phytopathogenic fungi (Fujino et al., 2025; Geisen et al., 2018).

For example, amoebae Acanthamoeba and Mayorella are capable of consuming spores of Fusarium culmorum — a dangerous pathogen of many crops (Fujino et al., 2025). The ciliate Colpoda is capable of consuming Ralstonia solanacearum — the causal agent of bacterial wilt of tomato (Fujino et al., 2025).

Indirect mechanism of pathogen suppression

The indirect mechanism consists in the fact that protists stimulate the activity of bacterial antagonists of phytopathogens. As already noted in section 3.1.4, protists create selective pressure that favors bacteria producing antibiotics and other protective compounds (Fujino et al., 2025; Geisen et al., 2018).

Studies have shown that in the presence of protists, bacteria Pseudomonas fluorescens produce more antibiotics that suppress the growth of phytopathogenic fungi (Fujino et al., 2025). This effect was confirmed under field conditions: the addition of organic fertilizers increased protist abundance, which in turn stimulated the activity of bacterial antagonists and reduced plant disease incidence (Fujino et al., 2025).

Examples of biocontrol involving protists

Several examples of successful biocontrol of phytopathogens involving protists have been described in the literature.

As Fujino et al. (2025) note, in the rhizosphere of tomatoes affected by Fusarium wilt, a negative correlation was observed between the abundance of predatory protists and pathogens. This indicates that protists naturally suppress pathogens in soil.

In a study by Guo et al. (2022), it was shown that in banana plantations with organic fertilization, the incidence of Fusarium wilt was lower than with mineral fertilizers. This was associated with higher abundance of predatory protists and bacterial antagonists in organic treatments (Fujino et al., 2025).

Studies in rice paddies showed a negative correlation between the abundance of predatory protists and Pythium sp. — a dangerous rice pathogen (Fujino et al., 2025).

Prospects for using protists as biocontrol agents

Despite promising results, the use of protists as biocontrol agents remains at the experimental stage. As Fujino et al. (2025) note, most studies were conducted under laboratory conditions, and only a few in the field.

Nevertheless, protists possess a number of advantages over bacteria and fungi as biocontrol agents. They are able to survive in soil in the form of cysts for a long time, are resistant to unfavorable conditions, and can be introduced into soil with organic fertilizers (Fujino et al., 2025; Geisen et al., 2018).

Why protists can be indicators of soil health

Protists possess a number of properties that make them convenient indicators of soil condition. As Geisen et al. (2018) note, these properties include:

• Short generation time (hours–days), allowing them to respond quickly to changing conditions;
• High diversity and functional significance;
• Sensitivity to environmental changes (moisture, temperature, pH, pollution);
• Possibility of quantitative assessment using molecular methods.

Composition and ratio of protist groups as indicators

Different groups of protists respond differently to changes in soil conditions. This makes it possible to use their composition and ratio as indicators of soil condition (Geisen et al., 2018).

For example, as Bonkowski et al. (2012) note, testate amoebae predominate in acidic soils, whereas naked amoebae and ciliates predominate in neutral soils. In soils with high organic matter content, the abundance of ciliates and flagellates is higher, whereas in poor soils, amoebae are more abundant (Bonkowski et al., 2012).

The ratio of predatory to bacterivorous protists may indicate the state of the food web: predominance of predators indicates maturity and stability of the community, whereas predominance of bacterivores indicates youth and instability (Geisen et al., 2018).

Using protists to assess the impact of agrotechnologies

Protists can be used to assess the impact of agrotechnologies on soil health. As Geisen et al. (2018) note, tillage, fertilizer application, and pesticides have a significant influence on protist communities.

Studies have shown that intensive tillage reduces the abundance and diversity of protists, especially large forms (Bonkowski et al., 2012). The application of organic fertilizers, by contrast, increases protist abundance and stimulates their activity (Fujino et al., 2025; Geisen et al., 2018).

Pesticides can exert both direct toxic effects on protists and indirect effects through changes in the abundance of their food (bacteria and fungi) (Geisen et al., 2018; Bonkowski et al., 2012).

Prospects for using protists as bioindicators

Despite promising prospects, the use of protists as bioindicators is still limited. As Geisen et al. (2018) note, the main obstacles include:

• Insufficient knowledge of the taxonomy and ecology of many groups;
• Lack of standardized assessment methods;
• High variability of communities in space and time.

Nevertheless, with the development of molecular methods and accumulation of data, protists may become an important tool for assessing soil health in agroecosystems (Geisen et al., 2018; Lentendu et al., 2022).

Interaction with fungi

Protists interact with fungi in diverse ways. As Geisen et al. (2018) note, many protists are mycophagous — they feed on fungal mycelium and spores.

Among mycophagous protists, amoebae (for example, Acanthamoeba, Leptomyxa) and ciliates (for example, Grossglockneria) are the most common (Geisen et al., 2018). These protists are capable of consuming a wide range of fungi, including phytopathogenic species (Fusarium, Pythium) (Fujino et al., 2025; Geisen et al., 2018).

The interaction of protists with fungi is not limited to predation. Some protists enter into symbiotic relationships with fungi, for example, forming lichens (Raven et al., 2013). Other protists, by contrast, are themselves parasites of fungi (Geisen et al., 2018).

Interaction with nematodes

Nematodes are one of the most numerous groups of soil animals. Protists interact with nematodes both as predators, consuming them, and as competitors for food (Bonkowski et al., 2012).

Large protists, such as testate amoebae and ciliates, are capable of consuming nematodes, especially small ones (Geisen et al., 2018). As Geisen et al. (2018) note, some testate amoebae use “pack hunting” to consume nematodes that are significantly larger than themselves.

In addition, protists and nematodes compete for bacterial food. As studies have shown, removal of amoebae from the soil food web led to an increase in the abundance of bacterivorous nematodes by 20–30% (Bonkowski et al., 2012). This indicates that protists and nematodes occupy similar ecological niches.

Interaction with microarthropods

Microarthropods (mites, springtails) are an important component of soil mesofauna. Protists interact with them as food and as competitors (Bonkowski et al., 2012).

Many microarthropods feed on protists, especially ciliates and amoebae. As Bonkowski et al. (2012) note, springtails and mites can consume significant numbers of protists, regulating their abundance.

In turn, protists can influence microarthropods through changes in the composition of bacterial and fungal communities that these animals feed on (Bonkowski et al., 2012; Geisen et al., 2018).

Protists as a link in the soil food web

Thus, protists occupy a central position in the soil food web. They consume bacteria and fungi (primary consumers of organic matter) and are themselves consumed by larger organisms — nematodes, microarthropods, and enchytraeids (Bonkowski et al., 2012; Geisen et al., 2018).

As Geisen et al. (2018) note, protists perform the role of “hub” organisms connecting microbial and animal components of the soil food web. Without them, energy and nutrient flows in soil would be substantially disrupted.

Taxonomic position

Plasmodiophora brassicae is one of the best-studied parasitic protists of plants. For a long time, this organism was regarded as a fungus and studied in mycology courses. However, modern molecular genetic studies have shown that P. brassicae belongs neither to true fungi (Eumycota) nor to oomycetes (Oomycota) (Webster & Weber, 2007; Alexopoulos, 1996).

According to modern classification, P. brassicae belongs to the supergroup Rhizaria, phylum Cercozoa, class Phytomyxea (Adl et al., 2012; Geisen et al., 2018). The closest relatives of P. brassicae are other phytomyxids — Spongospora, Polymyxa, Ligniera, as well as free-living cercozoans (Webster & Weber, 2007).

As Alexopoulos (1996) notes, phytomyxids (formerly known as plasmodiophoromycetes) are characterized by a unique type of nuclear division — the so-called “cruciform” mitosis, in which chromosomes are arranged in a ring around an elongated nucleolus. This feature is found neither in fungi nor in other groups of eukaryotes.

Biology and life cycle

Plasmodiophora brassicae is an obligate parasite that cannot grow and reproduce outside host plant cells. As Webster & Weber (2007) note, this organism infects mainly plants of the family Brassicaceae (crucifers), including cultivated species: cabbage, cauliflower, broccoli, turnip, radish, mustard, and rapeseed.

The life cycle of P. brassicae includes several stages (Webster & Weber, 2007; Alexopoulos, 1996):

Resting spores. In soil, resting spores can remain viable for 20 years or more. They are resistant to desiccation, temperature fluctuations, and chemical agents (Webster & Weber, 2007). The spores have a spiny surface and contain one haploid nuclear cell (Aist & Williams, 1971).

Germination and primary infection. In the presence of moisture and under the influence of substances released by roots of Brassicaceae plants (possibly allyl isothiocyanates), resting spores germinate, releasing motile zoospores with two flagella of unequal length (Macfarlane, 1970; Webster & Weber, 2007). Zoospores actively swim in the water film and attach to root hairs.

Penetration into the cell. The process of penetration of P. brassicae into the host plant cell was studied in detail by Aist and Williams (1971). The zoospore forms a cyst on the surface of the root hair, within which a tubular structure (Rohr) containing a pointed rod (Stachel) is formed. Upon contact with the plant cell wall, the Rohr everts, forming an adhesive structure (adhesorium), and the Stachel pierces the cell wall. The zoospore protoplast (amoeboid cell) penetrates into the host plant cell.

Primary plasmodium. Inside the root hair cell, the amoeba grows, divides, and turns into a primary (sporangial) plasmodium. This plasmodium is a multinucleate mass of cytoplasm lacking a cell wall, which feeds at the expense of the host cell (Webster & Weber, 2007). Nuclear divisions at this stage are cruciform (Alexopoulos, 1996).

Zoosporangia and secondary zoospores. The primary plasmodium breaks up into individual zoosporangia, each containing 4–8 secondary zoospores. Secondary zoospores are morphologically similar to primary ones and are capable of re-infecting root hairs, completing the asexual cycle (Webster & Weber, 2007).

Secondary plasmodium. After several cycles of asexual reproduction, the plasmodium migrates from the root hair into deeper root tissues, where it forms a secondary (sporogenous) plasmodium. This plasmodium undergoes meiotic divisions and forms resting spores (Garber & Aist, 1979; Webster & Weber, 2007).

Gall formation. As secondary plasmodia grow, host plant cells undergo hypertrophy (increase in size) and hyperplasia (enhanced division), which leads to the formation of characteristic galls on roots (Webster & Weber, 2007; Agrios, 2005).

Symptoms and harmfulness

Clubroot of cabbage is one of the most dangerous diseases of cruciferous crops. As Agrios (2005) notes, yield losses from clubroot can reach 50–100% in heavily infested fields.

Aboveground symptoms. The first symptoms of the disease appear as wilting of leaves in hot weather, although plants may recover at night. Gradually, plant growth slows, leaves turn yellow and wilt. Young plants may die soon after infection (Agrios, 2005; Webster & Weber, 2007).

Root symptoms. The most characteristic symptoms of clubroot appear on roots. Swellings (galls) form, which may be single or merge, covering the entire root system. Galls have a spindle-shaped, spherical, or lumpy form (Agrios, 2005). Old galls decompose under the action of bacteria and other microorganisms, releasing resting spores into the soil.

Effect on plant physiology. As Webster & Weber (2007) note, infection by P. brassicae leads to restructuring of plant metabolism: the content of auxins increases (1.7-fold) and cytokinins increases (2–3-fold) in gall tissues. This causes enhanced cell growth and gall formation, which become a new “sink” for photosynthetic products. As a result, the aboveground part of the plant experiences a shortage of nutrients, leading to growth suppression and reduced yield.

Control measures

Control of clubroot of cabbage is significantly difficult due to the long persistence of resting spores in soil and the wide range of host plants. As Webster & Weber (2007) note, resting spores of P. brassicae can persist in soil for up to 20 years, so short-term crop rotation is ineffective.

Crop rotation. Long-term crop rotation excluding cruciferous crops for 5–7 years or more reduces the number of resting spores in soil. However, even after a long break, spores may remain viable (Agrios, 2005; Webster & Weber, 2007).

Liming. Application of lime to raise soil pH to 7.2 and above is one of the most effective methods of clubroot control. Under neutral or slightly alkaline conditions, germination of resting spores is suppressed (Agrios, 2005; Webster & Weber, 2007). In addition, it has been shown that the addition of boron (10–20 mg/kg soil) in combination with high pH suppresses both primary and secondary infection (Webster & Weber, 2007).

Resistant varieties. Breeding resistant varieties is the most promising direction in clubroot control. However, as Webster & Weber (2007) note, resistance is often determined by a single gene and can be overcome by new pathogen races. By 1975, 34 physiological races of P. brassicae had been described (Buczacki et al., 1975). In recent years, new virulent pathotypes that overcome the resistance of commercial rapeseed varieties have been identified in Canada (Tso et al., 2021).

Agronomic measures. Additional control measures include growing seedlings in sterilized soil, improving drainage, destroying weeds of the Brassicaceae family (for example, shepherd’s purse), and disinfecting agricultural machinery (Agrios, 2005; Webster & Weber, 2007).

Taxonomic position and biology

Spongospora subterranea is the second representative of phytomyxids with important agronomic significance. This organism belongs to the same class Phytomyxea as P. brassicae (Webster & Weber, 2007; Adl et al., 2012).

S. subterranea causes powdery scab of potato — a disease affecting potato tubers. As Webster & Weber (2007) note, this parasite can also infect roots of tomatoes and other plants of the family Solanaceae.

The life cycle of S. subterranea is similar to that of P. brassicae and includes the formation of resting spores, zoospores, plasmodia, and zoosporangia. A characteristic feature is the formation of resting spores in the form of hollow balls (sporosori), which gave the genus its name — Spongospora (“spongy spore”) (Webster & Weber, 2007).

Symptoms and harmfulness

Symptoms on tubers. Powdery scab appears as small pustules (tubercles) on the surface of potato tubers. Initially, the pustules are light, then darken and turn into powdery lesions containing masses of resting spores (Webster & Weber, 2007). Affected tubers lose marketable appearance and store poorly.

Symptoms on roots. On potato roots, S. subterranea causes the formation of galls similar to those in clubroot of cabbage, but smaller (Webster & Weber, 2007).

Harmfulness. Powdery scab reduces potato yield and quality. In addition, S. subterranea is a vector of potato mop-top virus, which significantly increases the harmfulness of this organism (Webster & Weber, 2007; Draycott, 2006).

Control measures

Control measures for powdery scab are similar to those for clubroot of cabbage: crop rotation, use of healthy planting material, and improved drainage (Webster & Weber, 2007). However, as Webster & Weber (2007) note, transgenic potato plants containing the coat protein gene of potato mop-top virus showed complete resistance to the virus, which opens prospects for controlling this disease.

Taxonomic position and biology

The genus Polymyxa includes two species of important agronomic significance: P. betae and P. graminis. Both species belong to phytomyxids (class Phytomyxea) and are obligate plant parasites (Webster & Weber, 2007; Draycott, 2006).

Polymyxa betae infects plants of the family Chenopodiaceae, including sugar beet, spinach, and some weeds (Draycott, 2006). P. graminis infects cereal crops, including wheat, barley, rice, and maize (Webster & Weber, 2007).

The life cycle of Polymyxa is similar to that of other phytomyxids. Resting spores (cystospores) germinate, releasing zoospores that infect root cells. Plasmodia form in the cells and then transform into zoosporangia or resting spores (Draycott, 2006; Webster & Weber, 2007).

Role as virus vectors

The greatest agronomic importance of Polymyxa spp. is that they are vectors of dangerous plant viruses.

Polymyxa betae and rhizomania of beet. P. betae transmits Beet necrotic yellow vein virus (BNYVV) — the causal agent of rhizomania, one of the most dangerous diseases of sugar beet (Draycott, 2006). The virus persists in resting spores of P. betae and is transmitted by zoospores during root infection. Rhizomania can reduce sugar beet yield by 50–60% or more (Draycott, 2006).

Polymyxa graminis and cereal viruses. P. graminis transmits several viruses of cereal crops, including Wheat mosaic virus, Barley stripe mosaic virus, and others (Webster & Weber, 2007). These viruses can cause significant yield losses in cereals.

Control measures

Control of Polymyxa spp. and the viruses they transmit is significantly difficult because resting spores can persist in soil for many years (Draycott, 2006).

Crop rotation. Long-term crop rotation excluding susceptible crops reduces the number of resting spores in soil.

Resistant varieties. Breeding sugar beet varieties resistant to rhizomania is the most promising direction. However, as Draycott (2006) notes, resistance is often determined by a single gene (Rz) and can be overcome by new virus pathotypes.

Agronomic measures. Early sowing, good drainage, and avoidance of excessive soil moisture reduce the risk of infection (Draycott, 2006).

Olpidium

Olpidium is a genus of parasitic protists that for a long time was regarded as a representative of chytridiomycetes (Chytridiomycota), but modern studies have shown that this organism is closer to other groups of protists (Webster & Weber, 2007).

Olpidium brassicae parasitizes roots of cabbage and other plants, causing the formation of small galls. However, Olpidium is most important as a vector of viruses, including Tobacco necrosis virus and Lettuce big vein virus (Agrios, 2005; Webster & Weber, 2007).

Ligniera

Ligniera is a genus of phytomyxids parasitizing roots of various plants. These organisms cause the formation of small galls and can damage agricultural crops, although their economic importance is less than that of Plasmodiophora or Spongospora (Webster & Weber, 2007).

Other phytomyxids

In addition to those listed, phytomyxids include the genera Sorodiscus, Woronina, Octomyxa, and others. Most of them parasitize aquatic plants or algae and have no significant agronomic importance (Alexopoulos, 1996; Webster & Weber, 2007).

Cell wall

Fungi (Eumycota). The cell wall contains chitin and glucans. Chitin is a polymer of N-acetylglucosamine that gives strength and resistance to enzymatic decomposition (Agrios, 2005).

Oomycetes (Oomycota). The cell wall contains cellulose and glucans but does not contain chitin. This brings oomycetes closer to brown algae and other Stramenopiles (Agrios, 2005).

Phytomyxids (Phytomyxea). At the vegetative stage (plasmodium), the cell wall is absent. The plasmodium is surrounded only by a plasma membrane and is in direct contact with the cytoplasm of the host cell (Webster & Weber, 2007; Alexopoulos, 1996). Resting spores have a thick wall containing chitin (Moxham & Buczacki, 1983).

Nutrition

Fungi. Fungi are osmotrophs: they absorb dissolved nutrients through the cell wall. Many fungi secrete enzymes that destroy plant cell walls and feed on hydrolysis products (Agrios, 2005).

Oomycetes. Oomycetes are also osmotrophs, but their enzymatic apparatus differs from that of fungi (Agrios, 2005).

Phytomyxids. Phytomyxids are phagotrophs: they absorb nutrients by phagocytosis, engulfing portions of the host cell cytoplasm. As Webster & Weber (2007) note, plasmodia of P. brassicae are capable of forming pseudopodia and engulfing parts of the host cytoplasm.

Life cycles

Fungi. Fungal life cycles are diverse and include both asexual and sexual stages. Many fungi form mycelium — a network of hyphae that grow and branch (Agrios, 2005).

Oomycetes. Oomycetes form coenocytic mycelium and reproduce by zoospores and oospores. The sexual process is oogamy (Agrios, 2005).

Phytomyxids. Life cycles of phytomyxids include the formation of plasmodia (multinucleate masses of cytoplasm), zoospores, and resting spores. The sexual process is not fully understood, but it is assumed to be associated with the formation of resting spores (Webster & Weber, 2007; Alexopoulos, 1996).

Genetic and molecular features

Fungi. Fungi are eukaryotes with a true nucleus. Their cells may contain one or several nuclei. Genetic markers (18S rRNA, ITS) are used for identification and phylogenetic analysis (Agrios, 2005).

Oomycetes. Oomycetes are eukaryotes, but their genetic sequences (18S rRNA) differ significantly from those of fungi, confirming their belonging to a separate evolutionary lineage — Stramenopiles (Agrios, 2005).

Phytomyxids. Phytomyxids are eukaryotes with a unique type of nuclear division (cruciform mitosis). Their 18S rRNA sequences place them closer to Cercozoa (Rhizaria) than to fungi or oomycetes (Adl et al., 2012; Webster & Weber, 2007).

Summary table of differences

General characteristics of phototrophic protists

Phototrophic protists are organisms capable of photosynthesis, that is, using the energy of sunlight to synthesize organic compounds from carbon dioxide and water. In soils, phototrophic protists are represented by several groups: green algae, diatoms, euglenoids, and cyanobacteria (Raven et al., 2013; Geisen et al., 2018).

It is important to emphasize that cyanobacteria, although traditionally considered together with algae, are prokaryotes and do not belong to protists. However, in the context of soil ecology, they are often discussed together with phototrophic protists, since they occupy similar ecological niches and perform similar functions (Weil, 2017; Raven et al., 2013).

Phototrophic protists in soils are confined mainly to the upper horizons, where sufficient light penetrates for photosynthesis. As Geisen et al. (2018) note, in some soils photosynthetic protists can constitute a significant part of total protist diversity, although their contribution to total biomass is usually small.

Soil algae

Green algae (Chlorophyta) are the most diverse group of soil algae. They contain chlorophylls a and b, as well as accessory pigments, and store starch as a reserve nutrient (Raven et al., 2013). In soils, green algae are represented by unicellular and colonial forms.

The most common genera of soil green algae are Chlamydomonas, Chlorella, Scenedesmus, Stichococcus, and Chlorococcum (Raven et al., 2013; Geisen et al., 2018). These organisms are capable of rapid reproduction in the presence of moisture and light, forming characteristic green coatings on the soil surface.

Diatoms (Bacillariophyceae) are the second most important group of soil algae. Their distinctive feature is a siliceous frustule consisting of two valves (Raven et al., 2013). In soils, diatoms occur mainly in moist habitats, where they can form biological crusts.

As Geisen et al. (2018) note, diatoms play an important role in the silicon cycle in soils. Their siliceous frustules persist in soil for a long time after cell death and can be used for paleoecological reconstructions.

Euglenoids (Euglenoidea) are a group of flagellates among which both phototrophic and heterotrophic forms occur. Phototrophic euglenoids contain chlorophylls a and b and are capable of photosynthesis, but in the absence of light they can switch to heterotrophic nutrition (Ruppert et al., 2008; Raven et al., 2013). In soils, euglenoids are less common than green algae, but they can be found in moist habitats.

Cyanobacteria

Cyanobacteria are prokaryotic photosynthetic organisms that were traditionally regarded as “blue-green algae” (Raven et al., 2013; Weil, 2017). Cyanobacteria contain chlorophyll a and phycobiliproteins, which give them a characteristic blue-green color.

Cyanobacteria are widespread in soils, especially in waterlogged habitats such as rice paddies, bogs, and wet meadows (Weil, 2017). They are capable of fixing atmospheric nitrogen, which makes them an important component of the nitrogen cycle in agroecosystems.

As Weil (2017) notes, in rice paddies cyanobacteria can fix from 2 to 40 kg of nitrogen per hectare per year, which constitutes a significant part of rice nitrogen nutrition. Some cyanobacteria form symbiotic associations with plants, for example, with the aquatic fern Azolla, which is used as a biofertilizer in Southeast Asian countries (Weil, 2017).

In arid and semiarid regions, cyanobacteria are an important component of biological soil crusts (biocrusts). As Weil (2017) notes, these crusts consist of cyanobacteria, algae, fungi, mosses, and bacteria, which together form a complex community. Biocrusts protect soil from erosion, increase water infiltration, and enrich soil with nitrogen.

Role of phototrophic protists in agroecosystems

Phototrophic protists play an important role in agroecosystems, although their contribution to total soil biomass is usually small. As Geisen et al. (2018) note, photosynthetic protists contribute to the primary production of soil ecosystems, especially in arid and semiarid regions.

In rice paddies and other waterlogged agroecosystems, cyanobacteria fix atmospheric nitrogen, reducing the need for nitrogen fertilizers (Weil, 2017). In addition, cyanobacteria and algae release polysaccharides that promote aggregation of soil particles and improve soil structure (Weil, 2017).

However, mass reproduction of phototrophic protists in water bodies can lead to water “blooms,” which negatively affects water quality and can cause fish kills. In agroecosystems, this phenomenon is often associated with eutrophication — excessive input of biogenic elements (nitrogen and phosphorus) with fertilizers (Weil, 2017; Raven et al., 2013).

General characteristics of saprotrophic protists

Saprotrophic protists are organisms that feed on dead organic matter, decomposing it with enzymes and absorbing dissolved decomposition products. This type of nutrition is called osmotrophy (from Greek osmos — absorption, trophe — nutrition) (Hickman et al., 2004; Geisen et al., 2018).

Saprotrophic protists play an important role in the decomposition of organic matter in soils and water bodies. They are capable of decomposing a wide range of organic compounds, including cellulose, chitin, proteins, lipids, and others (Geisen et al., 2018).

Main groups of saprotrophic protists

Among saprotrophic protists, oomycetes (Oomycota) are the most common; they were traditionally regarded as fungi but belong to Stramenopiles (Adl et al., 2012; Geisen et al., 2018). Oomycetes such as Saprolegnia, Achlya, and Pythium are active decomposers of organic matter in aquatic and soil ecosystems.

As Geisen et al. (2018) note, many oomycetes are free-living saprotrophs that decompose organic matter using extracellular enzymes. Other oomycetes are facultative or obligate parasites of plants and animals.

In addition to oomycetes, saprotrophic protists include some groups of flagellates (for example, Bodo, Cercomonas), which are capable of both phagocytosis of bacteria and osmotrophy (Geisen et al., 2018; Bonkowski et al., 2012).

Role of saprotrophic protists in organic matter decomposition

Saprotrophic protists make a significant contribution to the decomposition of organic matter in soils and water bodies. As Geisen et al. (2018) note, oomycetes and other saprotrophic protists are capable of decomposing complex organic compounds that are not decomposed by bacteria.

In aquatic ecosystems, saprotrophic protists play a key role in the decomposition of plant remains, dead animals, and other organic materials (Geisen et al., 2018). In soils, their role is less studied but probably also significant, especially in moist habitats.

Saprotrophic protists release extracellular enzymes that break down complex organic molecules into simple compounds available for absorption. These compounds are then used by the protists themselves or by other microorganisms (Geisen et al., 2018).

Interaction with other microorganisms

Saprotrophic protists interact with other microorganisms, primarily bacteria and fungi, in the process of organic matter decomposition. As Geisen et al. (2018) note, saprotrophic protists can compete with bacteria for simple organic compounds, but can also promote the decomposition of complex compounds unavailable to bacteria.

In addition, saprotrophic protists can be consumed by other organisms, transferring organic matter along the food chain. Thus, they are an important link in the flow of energy and nutrients in ecosystems (Geisen et al., 2018; Bonkowski et al., 2012).

Lichens

Lichens are symbiotic associations of fungi and photosynthetic organisms (algae or cyanobacteria). In lichens, the fungus provides protection and absorption of water and minerals, while the photosynthetic partner supplies organic substances synthesized during photosynthesis (Raven et al., 2013; Weil, 2017).

Strictly speaking, lichens are not protists, since their fungal component belongs to Fungi, and the photosynthetic component can be either a protist (alga) or a prokaryote (cyanobacterium). However, in the context of soil ecology, lichens are often considered together with protists, since their photosynthetic component can be a protist (Raven et al., 2013).

Lichens play an important role in soil formation, especially on primary substrates such as rocks and volcanic rocks. They release organic acids that destroy minerals and promote accumulation of organic matter (Raven et al., 2013; Weil, 2017).

In agroecosystems, lichens are less common than in natural habitats, since they are sensitive to pollution and mechanical disturbance. However, they can be used as indicators of environmental condition (Raven et al., 2013).

Mixotrophic protists

Mixotrophic protists are organisms that combine photosynthesis with heterotrophic nutrition (phagocytosis or osmotrophy). As Geisen et al. (2018) note, mixotrophy is widespread among protists and allows them to survive under conditions when photosynthesis is impossible or insufficient.

Examples of mixotrophic protists are some dinoflagellates (for example, Gymnodinium, Ceratium), which are capable of both photosynthesis and phagocytosis (Ruppert et al., 2008; Geisen et al., 2018). In soils, mixotrophic protists are less common than in water bodies, but they can be found in moist habitats.

Mixotrophy gives protists an advantage under conditions of variable illumination and nutrient availability. As Geisen et al. (2018) note, in peat soils mixotrophic protists make a significant contribution to carbon sequestration.

Other symbiotic associations

In addition to lichens, protists form symbiotic associations with other organisms. For example, some ciliates and flagellates live in the intestines of termites and other animals, where they help the host digest cellulose (Ruppert et al., 2008; Hickman et al., 2004).

Other protists are endosymbionts of invertebrates, for example, zooxanthellae (dinoflagellates) in coral tissues (Ruppert et al., 2008). These symbiotic associations play an important role in ecosystem functioning, although their significance in agroecosystems is less studied.

Coccidia

Coccidia (Coccidia) are a group of apicomplexans (Apicomplexa) that parasitize intestinal cells of animals. The best-known genera are Eimeria, Isospora, Cryptosporidium, and Toxoplasma (Hickman et al., 2004; Ruppert et al., 2008).

Coccidia cause coccidiosis, which manifests as diarrhea, dehydration, and weight loss. In farm animals, coccidiosis can lead to significant economic losses (Hickman et al., 2004; Ruppert et al., 2008).

As Hickman et al. (2004) note, Eimeria is one of the most important genera of coccidia in veterinary terms. Different species of Eimeria parasitize different animals: E. tenella in chickens, E. bovis in cattle, and E. suis in pigs. Coccidiosis in chicks can cause mass mortality, especially under crowded housing conditions.

The life cycle of coccidia includes stages of schizogony (multiple division), gametogony (formation of gametes), and sporogony (formation of oocysts). Oocysts are excreted with feces and can persist in soil for a long time, infecting new hosts (Hickman et al., 2004; Ruppert et al., 2008).

Piroplasms

Piroplasms (Piroplasmida) are a group of apicomplexans that parasitize erythrocytes of animals. The best-known genera are Babesia and Theileria (Ruppert et al., 2008; Hickman et al., 2004).

Piroplasms are transmitted through tick bites and cause diseases known as piroplasmoses or babesioses. In cattle, babesiosis manifests as fever, anemia, jaundice, and can lead to animal death (Ruppert et al., 2008; Hickman et al., 2004).

As Ruppert et al. (2008) note, piroplasms have a complex life cycle involving alternation of hosts (vertebrates and ticks). In the vertebrate body, the parasite reproduces in erythrocytes, causing their destruction. In the tick body, the parasite undergoes a sexual cycle and is transmitted to the next generation of ticks through eggs.

Veterinary importance

Coccidia and piroplasms are of important veterinary significance, since they cause diseases of farm animals and pets. As Hickman et al. (2004) and Ruppert et al. (2008) note, these diseases can lead to significant economic losses in livestock production.

Control measures for coccidiosis include the use of coccidiostats (drugs suppressing coccidia development), maintenance of animal hygiene, and preventive treatments. Control of piroplasmoses includes destruction of tick vectors and use of antiparasitic drugs (Hickman et al., 2004; Ruppert et al., 2008).

Giardia duodenalis (giardia)

Giardia duodenalis (also known as G. lamblia or G. intestinalis) is a flagellate parasitizing the small intestine of humans and other mammals. This parasite causes giardiasis, which manifests as diarrhea, bloating, nausea, and general malaise (Hickman et al., 2004; Ruppert et al., 2008).

As Hickman et al. (2004) note, Giardia has a characteristic pear-shaped form with two nuclei and eight flagella. The parasite attaches to the intestinal wall with a special disc and feeds at the expense of the host.

Cysts of Giardia are excreted with feces and can persist in soil and water for several weeks or even months. Infection occurs through consumption of contaminated water or food, as well as contact with contaminated surfaces (Hickman et al., 2004; Bonkowski et al., 2012).

Giardiasis is widespread throughout the world, especially in countries with low sanitation levels. In developed countries, giardiasis outbreaks are often associated with contamination of tap water or water bodies used for swimming (Hickman et al., 2004).

Entamoeba histolytica (dysentery amoeba)

Entamoeba histolytica is an amoeba parasitizing the large intestine of humans. This parasite causes amoebic dysentery — a disease that manifests as bloody diarrhea, abdominal pain, and fever (Hickman et al., 2004; Ruppert et al., 2008).

As Hickman et al. (2004) note, E. histolytica can penetrate the intestinal wall, causing ulcer formation. In severe cases, the parasite can spread to the liver and other organs, causing abscesses.

Cysts of E. histolytica are excreted with feces and can persist in soil and water. Infection occurs through consumption of contaminated water or food. Amoebic dysentery is widespread in tropical and subtropical regions where sanitary conditions are poor (Hickman et al., 2004; Ruppert et al., 2008).

Toxoplasma gondii (toxoplasma)

Toxoplasma gondii is an apicomplexan parasitizing cells of various tissues of humans and animals. This parasite causes toxoplasmosis, which in healthy people is usually asymptomatic but can be dangerous for pregnant women and immunocompromised people (Hickman et al., 2004; Ruppert et al., 2008).

As Hickman et al. (2004) note, the definitive host of T. gondii is cats, in whose intestines the parasite undergoes the sexual cycle and forms oocysts. Oocysts are excreted with cat feces and can persist in soil for a long time.

Human infection occurs through consumption of insufficiently heat-treated meat containing tissue cysts, or through contact with soil contaminated with cat feces. Toxoplasmosis can cause serious complications in pregnant women (congenital toxoplasmosis) and in immunocompromised people (Hickman et al., 2004; Ruppert et al., 2008).

Other human parasites

In addition to those listed, protists parasitizing humans include:

• Cryptosporidium parvum — causes cryptosporidiosis, manifested as diarrhea. Oocysts can persist in soil and water and are resistant to chlorination (Hickman et al., 2004; Ruppert et al., 2008).

• Cyclospora cayetanensis — causes cyclosporiasis, characterized by diarrhea. Oocysts are excreted with feces and can contaminate soil and water (Hickman et al., 2004).

• Balantidium coli — a ciliate parasitizing the intestines of humans and pigs. Causes balantidiasis, manifested as diarrhea (Ruppert et al., 2008; Hickman et al., 2004).

• Naegleria fowleri — an amoeba inhabiting soil and water. Can cause primary amoebic meningoencephalitis — a rare but extremely dangerous disease (Bonkowski et al., 2012; Geisen et al., 2018).

• Acanthamoeba spp. — amoebae widespread in soil and water. Can cause keratitis (inflammation of the cornea) and granulomatous amoebic encephalitis in immunocompromised people (Bonkowski et al., 2012; Geisen et al., 2018).

Sanitary importance and preventive measures

Parasitic protists of humans are of important sanitary significance, especially in the context of soil and water contamination. As Bonkowski et al. (2012) note, many parasitic protists can persist in soil in the form of cysts or oocysts for a long time and infect people through contact with contaminated soil or consumption of contaminated products.

Preventive measures include observance of personal hygiene rules, use of clean water, thorough heat treatment of meat and vegetables, and protection of soil and water from fecal contamination (Bonkowski et al., 2012; Hickman et al., 2004).

Particular attention should be paid to the sanitary condition of soil in agroecosystems, since contamination of soil with pathogenic protists can lead to infection of agricultural crops and animals, and through them humans (Bonkowski et al., 2012; Geisen et al., 2018).

Protists as “Trojan horses” for pathogenic bacteria

Of special attention is the role of free-living amoebae (for example, Acanthamoeba, Naegleria, Hartmannella) as reservoirs for pathogenic bacteria. As Bonkowski et al. (2012) and Geisen et al. (2018) note, bacteria such as Legionella, Chlamydia, Listeria, Salmonella, and Mycobacterium can survive and multiply inside amoeba cells.

This phenomenon has been called the “Trojan horse” of the microbial world: amoebae protect bacteria from unfavorable environmental conditions and disinfectants, and under certain conditions bacteria can leave the amoeba and infect humans (Bonkowski et al., 2012; Geisen et al., 2018).

As Geisen et al. (2018) note, coevolution of bacteria inside amoebae may have led to their acquiring the ability to overcome the human immune system. This makes the problem of interaction between protists and pathogenic bacteria important not only for ecology but also for medicine.

Problems in diagnosing protist diseases

Diagnosis of diseases caused by protists is significantly difficult, since the symptoms of these diseases are often similar to those of fungal and oomycete infections. As Agrios (2005) notes, clubroot of cabbage and powdery scab of potato can be mistakenly diagnosed as fungal diseases, leading to incorrect choice of control measures.

In addition, traditional diagnostic methods based on microscopy require high qualification and do not always allow distinguishing protists from fungi at early stages of infection. As Tso et al. (2021) note, Plasmodiophora brassicae is characterized by a long latent phase, when symptoms have not yet appeared but the pathogen is already present in the soil.

Molecular diagnostic methods

Modern molecular methods make it possible to diagnose protist diseases at early stages and with high accuracy. As Tso et al. (2021) note, PCR tests based on amplification of specific genome regions have been developed for diagnosing P. brassicae.

PCR diagnosis of clubroot of cabbage. The first PCR tests for diagnosing P. brassicae were developed in the late 1990s. As Tso et al. (2021) note, Ito et al. (1999) developed a nested PCR based on the isopentenyltransferase gene, and Faggian et al. (1999) based on the ITS region of ribosomal DNA. These tests can detect the pathogen in soil at a concentration of only 10³ resting spores per gram of soil (Tso et al., 2021).

Quantitative PCR (qPCR). For quantitative assessment of P. brassicae inoculum in soil, qPCR tests have been developed that allow determination of resting spore numbers. As Tso et al. (2021) note, these tests can be used to assess the risk of field infestation and choose resistant varieties.

Other methods. PCR tests based on amplification of specific genome regions have also been developed for diagnosing Spongospora subterranea (Tso et al., 2021; Draycott, 2006). For Polymyxa spp., multiplex PCR tests have been developed that allow simultaneous detection of several species (Draycott, 2006).

Pathotyping of protists

Determination of pathotypes (races) of protists is important for choosing resistant varieties and developing control strategies. As Tso et al. (2021) note, differential sets have been developed for P. brassicae that allow distinguishing pathotypes by their virulence on different plant varieties.

Differential sets. The classical Williams set (Williams, 1966) includes four differential hosts and allows distinguishing up to 16 pathotypes. The European ECD set (Buczacki et al., 1975) includes 15 hosts. The Canadian CCD set (Strelkov et al., 2018) includes 13 genotypes and allows distinguishing more pathotypes than previous sets (Tso et al., 2021).

Molecular pathotyping. As Tso et al. (2021) note, in recent years molecular methods for pathotyping P. brassicae based on DNA polymorphism analysis have been developed. These methods allow rapid and accurate determination of pathotypes without labor-intensive bioassays.

Problems of pathotyping. As Tso et al. (2021) note, existing differential sets are based on phenotypic traits (virulence), which do not always correspond to genetic differences. In addition, bioassays require a long time (6 weeks) and high labor costs.

Practical recommendations for diagnosis

For effective diagnosis of protist diseases, a comprehensive approach is recommended, including:

1. Visual inspection of plants for characteristic symptoms (galls on roots, pustules on tubers) (Agrios, 2005).

2. Microscopy to detect resting spores or plasmodia in plant tissues (Tso et al., 2021).

3. Molecular diagnostics (PCR, qPCR) to confirm the diagnosis and quantify inoculum (Tso et al., 2021).

4. Pathotyping to determine the pathogen race and choose resistant varieties (Tso et al., 2021).

Why protists can be indicators

Protists possess a number of properties that make them convenient indicators of soil condition (Geisen et al., 2018):

• Short generation time (hours–days) allows them to respond quickly to changing conditions.
• High diversity and functional significance.
• Sensitivity to environmental changes (moisture, temperature, pH, pollution).
• Possibility of quantitative assessment using molecular methods (Lentendu et al., 2022).

As Geisen et al. (2018) note, the composition and ratio of protist groups can indicate the state of the soil food web, organic matter level, pH, and other parameters.

Composition and ratio of groups as indicators

Different groups of protists respond differently to changes in soil conditions. This makes it possible to use them as indicators (Geisen et al., 2018; Bonkowski et al., 2012).

Soil acidity. In acidic soils, testate amoebae predominate, whereas in neutral soils, naked amoebae and ciliates predominate (Bonkowski et al., 2012). As Geisen et al. (2018) note, testate amoebae are especially numerous in bogs and peatlands with low pH.

Organic matter. In soils with high organic matter content, the abundance of ciliates and flagellates is higher, whereas in poor soils, amoebae are more abundant (Bonkowski et al., 2012).

Ratio of predatory and bacterivorous forms. Predominance of predatory protists indicates maturity and stability of the community, whereas predominance of bacterivores indicates youth and instability (Geisen et al., 2018).

Moisture. Protists are sensitive to soil moisture. As Geisen et al. (2018) note, under dry conditions the abundance and diversity of protists decrease, while under waterlogged conditions they increase.

Using protists to assess the impact of agrotechnologies

Protists can be used to assess the impact of agrotechnologies on soil health (Geisen et al., 2018; Bonkowski et al., 2012).

Tillage. Intensive tillage reduces the abundance and diversity of protists, especially large forms (Bonkowski et al., 2012). As Geisen et al. (2018) note, minimal tillage (no-till) helps preserve more diverse protist communities.

Fertilizers. Application of organic fertilizers increases protist abundance and stimulates their activity (Fujino et al., 2025; Geisen et al., 2018). Mineral fertilizers have a less pronounced effect but can change community composition.

Pesticides. Pesticides can exert both direct toxic effects on protists and indirect effects through changes in the abundance of their food (bacteria and fungi) (Geisen et al., 2018; Bonkowski et al., 2012).

Prospects for using protists as bioindicators

Despite promising prospects, the use of protists as bioindicators is still limited. As Geisen et al. (2018) note, the main obstacles include:

• Insufficient knowledge of the taxonomy and ecology of many groups.
• Lack of standardized assessment methods.
• High variability of communities in space and time.

Nevertheless, with the development of molecular methods (18S rRNA metabarcoding) and accumulation of data, protists may become an important tool for assessing soil health in agroecosystems (Geisen et al., 2018; Lentendu et al., 2022).

Direct mechanism of biocontrol

Predatory protists are capable of directly consuming phytopathogenic microorganisms, including fungi, oomycetes, and bacteria. As Fujino et al. (2025) note, this mechanism has been called the “direct” mechanism of biocontrol.

Examples of direct biocontrol. As Fujino et al. (2025) note, amoebae Acanthamoeba and Mayorella are capable of consuming spores of Fusarium culmorum — a dangerous pathogen of many crops. The ciliate Colpoda is capable of consuming Ralstonia solanacearum — the causal agent of bacterial wilt of tomato.

Effectiveness. As Fujino et al. (2025) note, the effectiveness of direct biocontrol depends on protist abundance, their food preferences, and pathogen density. Under laboratory conditions, protists can reduce pathogen abundance by 50–90%.

Indirect mechanism of biocontrol

The indirect mechanism consists in the fact that protists stimulate the activity of bacterial antagonists of phytopathogens. As already noted in lecture 3, protists create selective pressure that favors bacteria producing antibiotics and other protective compounds (Fujino et al., 2025; Geisen et al., 2018).

Examples of indirect biocontrol. As Fujino et al. (2025) note, in the presence of protists, bacteria Pseudomonas fluorescens produce more antibiotics that suppress the growth of phytopathogenic fungi. This effect was confirmed under field conditions: the addition of organic fertilizers increased protist abundance, which stimulated the activity of bacterial antagonists and reduced plant disease incidence.

Synergism with organic fertilizers. As Fujino et al. (2025) note, organic fertilizers increase protist abundance, which in turn stimulates the activity of bacterial antagonists. This synergism can be used to develop biocontrol strategies.

Prospects for using protists as biocontrol agents

Despite promising results, the use of protists as biocontrol agents remains at the experimental stage. As Fujino et al. (2025) note, most studies were conducted under laboratory conditions, and only a few in the field.

Advantages of protists. Protists possess a number of advantages over bacteria and fungi as biocontrol agents: they are able to survive in soil in the form of cysts for a long time, are resistant to unfavorable conditions, and can be introduced into soil with organic fertilizers (Fujino et al., 2025; Geisen et al., 2018).

Problems and limitations. As Fujino et al. (2025) note, the main problems include:

• Insufficient knowledge of protist specificity to pathogens.
• Difficulty in scaling up protist production.
• Lack of standardized application methods.

Prospects. As Fujino et al. (2025) and Geisen et al. (2018) note, further research should be aimed at:

• Screening protists effective against specific pathogens.
• Developing methods for mass cultivation of protists.
• Conducting field trials to assess biocontrol effectiveness.

Tillage

Tillage is one of the most significant factors affecting protist communities. As Bonkowski et al. (2012) note, intensive tillage (plowing, cultivation) destroys soil aggregates, changes pore space, and reduces the abundance and diversity of protists.

Impact on different groups. As Geisen et al. (2018) note, large protists (ciliates, testate amoebae) are more sensitive to tillage than small ones (flagellates, naked amoebae). In tilled soils, small forms predominate, capable of quickly colonizing disturbed habitats.

Comparison with no-till. As Bonkowski et al. (2012) note, in no-till systems the abundance and diversity of protists are higher than in traditional systems. This is due to preservation of soil structure and organic matter.

Fertilizers

Fertilizer application has a significant impact on protist communities. As Geisen et al. (2018) note, organic fertilizers (manure, compost) increase the abundance and diversity of protists, since they contain organic matter that stimulates bacterial growth — food for protists.

Organic fertilizers. As Fujino et al. (2025) note, organic fertilizers increase the abundance of predatory protists and stimulate their activity. This, in turn, stimulates the activity of bacterial antagonists and reduces plant disease incidence.

Mineral fertilizers. As Geisen et al. (2018) note, mineral fertilizers have a less pronounced effect on protists but can change community composition. High doses of nitrogen fertilizers can reduce protist diversity due to soil acidification.

Pesticides

Pesticides can exert both direct and indirect effects on protist communities. As Geisen et al. (2018) note, the direct toxic effect of pesticides on protists has been insufficiently studied, but some studies show that herbicides and insecticides can reduce the abundance and diversity of protists.

Indirect influence. As Bonkowski et al. (2012) note, pesticides can influence protists indirectly through changes in the abundance of their food (bacteria and fungi). For example, fungicides that suppress fungi can reduce the abundance of mycophagous protists.

Resistance to pesticides. As Geisen et al. (2018) note, some protists possess resistance to pesticides and can accumulate in treated soils. This can lead to disruption of food chains and reduction of the biocontrol potential of soil.

Crop rotation and cover crops

Crop rotation and cover crops have a significant impact on protist communities. As Geisen et al. (2018) note, plant diversity in crop rotation promotes protist diversity, since different plants release different root exudates that stimulate different groups of bacteria and fungi.

Cover crops. As Weil (2017) notes, cover crops (for example, rye, rapeseed, radish) increase the abundance and diversity of protists, especially in the rhizosphere. This is associated with the input of organic matter and stimulation of microbial activity.

Effect on biocontrol. As Fujino et al. (2025) note, cover crops can stimulate the biocontrol potential of soil by increasing the abundance of predatory protists and bacterial antagonists.

Protists and degradation of pollutants

Protists can participate in the degradation of organic pollutants (petroleum products, pesticides, industrial waste) both directly and indirectly (Geisen et al., 2018).

Direct degradation. As Geisen et al. (2018) note, some protists are capable of degrading organic pollutants using extracellular enzymes. For example, oomycetes and some flagellates are capable of degrading petroleum products and chlorinated hydrocarbons.

Indirect degradation. As Geisen et al. (2018) note, protists stimulate the activity of bacteria that degrade pollutants. By consuming bacteria, protists release nutrients that stimulate the growth of degrader bacteria.

Protists and heavy metals

Protists can accumulate heavy metals (lead, cadmium, copper, zinc) and thus participate in their detoxification. As Geisen et al. (2018) note, some protists (for example, testate amoebae) are capable of accumulating metals in their shells, which are then buried in soil.

Bioindication of pollution. As Geisen et al. (2018) note, the composition of protist communities can be used to indicate soil contamination with heavy metals. Some protist species are sensitive to metals, while others are resistant, which allows using them as bioindicators.

Protists and organic pollutants

Protists can participate in the degradation of organic pollutants, including pesticides and petroleum products. As Geisen et al. (2018) note, some protists are capable of degrading these compounds using extracellular enzymes.

Synergism with bacteria. As Geisen et al. (2018) note, protists and bacteria act synergistically in the degradation of organic pollutants. Protists consume bacteria, stimulating their growth and activity, and also release nutrients necessary for degradation.

Prospects for bioremediation using protists

Despite promising prospects, the use of protists in bioremediation remains at the experimental stage. As Geisen et al. (2018) note, the main problems include:

• Insufficient knowledge of the ability of protists to degrade specific pollutants.
• Difficulty in scaling up bioremediation processes.
• Lack of standardized methods for assessing effectiveness.

Prospects. As Geisen et al. (2018) note, further research should be aimed at:

• Screening protists effective in degrading specific pollutants.
• Studying the synergism of protists and bacteria in bioremediation.
• Developing methods for introducing protists into contaminated soils.

Light microscopy

Light microscopy is the traditional and most accessible method for studying protists. As Abraham et al. (2019) note, stereomicroscopes and microscopes with phase-contrast or differential interference contrast optics are used to observe living protists.

Observation of living cells. To study protist morphology, cells are placed on a microscope slide in a drop of water or nutrient medium. However, active movement of many protists complicates their observation. To slow movement, various agents are used: methylcellulose, polyox resin WSR, Protoslo (Abraham et al., 2019). These substances increase the viscosity of the medium and slow movement but can change cell shape and cause premortal changes, so their use is limited (Abraham et al., 2019).

Cell immobilization. A more preferable method is immobilization of cells using a micropipette and a coverslip with Vaseline “feet.” Cells are slightly compressed between the slide and coverslip, which allows observation of their morphology without chemical exposure (Abraham et al., 2019). Commercial microcompressors are also used (Skovorodkin, 1990).

Morphological characters. Under light microscopy, the following are taken into account: size, cell shape, presence and structure of the shell (in testate amoebae), arrangement of flagella or cilia, structure of the nuclear apparatus (in ciliates — macro- and micronuclei), presence and location of vacuoles, and digestive inclusions (Bonkowski et al., 2012; Ruppert et al., 2008).

Special staining methods

To reveal fine morphological details, various staining methods are used.

Silver impregnation. As Abraham et al. (2019) note, silver impregnation is the most widely used method for studying ciliate morphology. There are three main protocols: silver carbonate impregnation (Fernandez-Galiano, 1976), silver nitrate impregnation (Chatton & Lwoff, 1930), and silver proteinate (protargol) impregnation (Wilbert, 1975). These methods reveal the arrangement of kinetosomes, infraciliature, silver lines, and the nuclear apparatus (Abraham et al., 2019).

Protargol. Protargol (silver proteinate) is the most valuable method for ciliate taxonomy, since it reveals both ciliature and the nuclear apparatus (Abraham et al., 2019). However, protargol is no longer produced commercially and must be synthesized in the laboratory (Pan et al., 2013). The procedure includes fixation, staining in protargol solution, development, and mounting in balsam (Abraham et al., 2019).

FLUTAX staining. As Abraham et al. (2019) note, FLUTAX (a fluorescent derivative of taxol) binds to microtubules and allows visualization of ciliature and other microtubular structures. This method is faster and simpler than silver impregnation but does not yield permanent preparations (Arregui et al., 2003).

Feulgen staining. For selective detection of the nuclear apparatus, the Feulgen reaction is used, which stains DNA crimson (Abraham et al., 2019). This method allows study of the shape, size, and location of macro- and micronuclei.

DAPI staining. DAPI is a fluorescent dye that binds to DNA. It is used for rapid visualization of protist nuclei using epifluorescence microscopy (Abraham et al., 2019; Zhan et al., 2014). However, DAPI-stained preparations are not suitable for long-term storage.

FISH (fluorescence in situ hybridization). As Abraham et al. (2019) note, FISH allows identification of protists in complex samples using fluorescently labeled oligonucleotide probes that hybridize to target rRNA sequences. This method allows simultaneous identification of the taxon and study of its morphology (Fried et al., 2002; Zhan et al., 2014).

Electron microscopy

Electron microscopy (EM) allows study of the ultrastructure of protists, which is important for taxonomy and phylogeny.

Scanning electron microscopy (SEM). SEM provides a three-dimensional image of the cell surface and allows study of morphological details that are difficult to reveal by light microscopy (Abraham et al., 2019). The procedure includes fixation in a mixture of OsO₄ and HgCl₂, dehydration, critical point drying, and gold coating (Abraham et al., 2019). SEM is used to study the shells of testate amoebae, diatom frustules, and the surface of ciliates.

Transmission electron microscopy (TEM). TEM allows study of the internal ultrastructure of the cell: structure of mitochondria, chloroplasts, nuclear envelope, flagellar apparatus, kinetosomes, and alveoli (Ruppert et al., 2008; Adl et al., 2012). Ultrastructural characters are widely used in modern protist taxonomy (Adl et al., 2012; Geisen et al., 2018).

Isolation and cultivation of protists

Cultivation of protists allows obtaining pure cultures for detailed study of morphology, physiology, and genetics. As Bonkowski et al. (2012) note, dilution, enrichment, and cloning methods are used to isolate protists from soil.

Serial dilution method. Soil suspension is sequentially diluted in sterile water or nutrient medium, then inoculated into wells of 96-well plates. As Lentendu et al. (2022) note, amoeba saline solution (Page, 1988) with the addition of Escherichia coli as food is used for culturing protists. Optimal conditions: 50 µl of a 1:1000 dilution of soil suspension and 100 µl of amoeba solution with E. coli cells (10⁷ cells/ml) (Lentendu et al., 2022).

Migration method. To separate different types of protists, the migration method (Neff, 1958) is used, based on the ability of protists to actively move through agar bridges (Lentendu et al., 2022).

Cloning. To obtain axenic (pure) cultures, antibiotics are used (Jones et al., 1973). However, most soil protists cannot be cultivated on standard media (Geisen et al., 2018).

Most Probable Number (MPN) method

The Most Probable Number (MPN) method is a quantitative method for estimating protist abundance in soil. As Bonkowski et al. (2012) note, MPN is based on statistical estimation of the number of viable cells from the results of their growth in serial dilutions.

MPN is the only practical quantitative method for organisms occurring in soil at concentrations of 10³–10⁴ cells/g (Bonkowski et al., 2012). However, the method has disadvantages: it is labor-intensive, requires a long time (up to several weeks), and gives biased estimates (Adl, 2003).

Limitations of culture methods

Culture methods have a number of limitations:

• Most soil protists do not grow on standard media (Geisen et al., 2018).
• Cultivation requires significant time and labor.
• Some protists (for example, obligate parasites) cannot be cultured outside the host organism (Webster & Weber, 2007).
• Cultivation results may not reflect the real diversity of the community (Lentendu et al., 2022).

DNA extraction

Molecular methods begin with extraction of total DNA from soil or from pure protist cultures. As Lentendu et al. (2022) note, commercial kits such as DNeasy PowerSoil (Qiagen) or NucleoSpin Soil (MACHEREY-NAGEL) are used for DNA extraction from soil. Usually 0.25–0.3 g of soil is used (Lentendu et al., 2022).

PCR and marker genes

Various marker genes are used for protist identification. As Lentendu et al. (2022) note, the 18S rRNA gene is most widely used, as it contains both conserved and variable regions.

18S rRNA gene. This is the most popular marker for protist metabarcoding. As Lentendu et al. (2022) note, hypervariable regions V4 and V9 are used. The V4 region (about 380–420 bp) provides more phylogenetic information but has greater length variability. The V9 region (about 120–150 bp) is shorter, making it convenient for sequencing on short-read platforms (Illumina MiSeq 2×150 bp), but it is less informative (Lentendu et al., 2022).

Primers. For amplification of the V4 region, primers TAReuk454FWD1 and TAReukREV3 are used (Stoeck et al., 2010; Lentendu et al., 2022). For the V9 region, primers 1389F and 1510R are used (Amaral-Zettler et al., 2009). For specific groups (for example, cercozoans, ciliates), group-specific primers have been developed (Lentendu et al., 2022).

ITS and COI. For some protist groups, ITS regions (internal transcribed spacers) and the COI gene (cytochrome oxidase I) are used. As Lentendu et al. (2022) note, ITS is used for phylogenetic studies at the species level, and COI for some groups (for example, diatoms).

Metabarcoding and high-throughput sequencing

Metabarcoding is a method based on the amplification and sequencing of a marker gene from total DNA extracted from soil. As noted by Lentendu et al. (2022), this approach makes it possible to identify hundreds and thousands of operational taxonomic units (OTUs) or amplicon sequence variants (ASVs) in a single sample.

Stages of metabarcoding. As noted by Lentendu et al. (2022), the process includes:

1. DNA extraction from soil.

2. PCR amplification of the marker gene using barcoded primers.

3. Purification and quantification of PCR products.

4. Pooling of samples in equimolar concentrations.

5. Sequencing on the Illumina MiSeq platform (2×300 bp or 2×250 bp).

6. Bioinformatics processing: filtering, removal of primers and barcodes, ASV clustering, taxonomic identification (Lentendu et al., 2022).

Bioinformatics tools. As noted by Lentendu et al. (2022), Cutadapt (Martin, 2011), VSEARCH (Rognes et al., 2016), and DADA2 (Callahan et al., 2016) are used for data processing. DADA2 allows distinguishing ASVs with single-nucleotide resolution, which is important for identifying cryptic species (Lentendu et al., 2022).

Reference databases

Specialized databases are used for taxonomic identification of sequences. As noted by Lentendu et al. (2022), for protists the most important are:

• PR² (Protist Ribosomal Reference database) — a curated database of 18S rRNA sequences of protists (Guillou et al., 2013).
• SILVA — a comprehensive database of ribosomal RNAs for all domains of life, including protists (Quast et al., 2012). SILVA contains more than 3 million SSU and LSU rRNA sequences (Quast et al., 2012).
• MIDORI — a database of mitochondrial sequences for animal metabarcoding (Leray et al., 2022).

Quantitative molecular methods

qPCR (quantitative PCR). As noted by Tso et al. (2021), qPCR makes it possible to quantify the abundance of pathogenic protists in soil. For Plasmodiophora brassicae, qPCR tests based on TaqMan probes have been developed (Wallenhammar et al., 2012; Deora et al., 2015).

ddPCR (droplet digital PCR). As noted by Tso et al. (2021), ddPCR is a more accurate quantification method, less sensitive to PCR inhibitors. It has been used for quantification of P. brassicae in soil (Wen et al., 2020).

PMA-qPCR. To distinguish viable and non-viable cells, PMA (propidium monoazide) is used in combination with qPCR. PMA penetrates cells with damaged membranes and binds to DNA, preventing its amplification (Al-Daoud et al., 2016; Tso et al., 2021).

FISH and other in situ methods

FISH (fluorescence in situ hybridization). As noted by Abraham et al. (2019), FISH makes it possible to identify protists in complex samples using fluorescently labeled probes. This method allows simultaneous identification of the taxon and study of its morphology (Fried et al., 2002; Zhan et al., 2014).

Cryptic species

One of the main problems in protist identification is the presence of cryptic species — morphologically indistinguishable but genetically and ecologically distinct forms. As noted by Geisen et al. (2018), molecular methods reveal a huge number of cryptic species among protists.

For example, as noted by Lentendu et al. (2022), the diversity of 18S rDNA genotypes within morphospecies of cercomonads can be very high. Many genotypes have cosmopolitan distribution, but some are ecologically or geographically restricted (Bass et al., 2007; Lentendu et al., 2022).

Polymorphism and life cycles

Many protists have complex life cycles with several morphologically distinct stages. As noted by Geisen et al. (2018), this complicates identification, since different stages of one species may be described as different species.

For example, in myxomycetes the plasmodial and spore-forming stages are morphologically completely different (Ruppert et al., 2008). In apicomplexans, different stages of the life cycle are also morphologically distinct (Hickman et al., 2004).

Incompleteness of reference databases

As noted by Lentendu et al. (2022), many sequences obtained from the environment have no close matches in reference databases. This complicates taxonomic identification and leads to a significant proportion of sequences remaining unclassified (Geisen et al., 2018; Lentendu et al., 2022).

Primer bias

Different primers amplify different groups of protists differently. As noted by Lentendu et al. (2022), universal primers may fail to amplify some groups, leading to underestimation of their diversity. Conversely, some groups may be preferentially amplified, leading to overestimation of their abundance (Lentendu et al., 2022; Geisen et al., 2018).

Introns and length variability

The 18S rRNA gene may contain introns that disrupt amplification and sequence analysis. As noted by Lentendu et al. (2022), in some testate amoebae (for example, Difflugia bacillarium) the V9 region contains a 427 bp intron, and in glissomonads — an intron of about 600 bp (Gomaa et al., 2012; Ekelund et al., 2004).

Quantitative limitations

Metabarcoding gives relative, not absolute, estimates of abundance. As noted by Lentendu et al. (2022), the number of reads is not always proportional to cell numbers because of differences in rRNA gene copy number, amplification efficiency, and other factors.

Need to combine methods

As noted by Abraham et al. (2019), reliable identification of protists requires an integrative approach combining morphological and molecular data. This is especially important for identifying cryptic species and understanding evolutionary relationships (Geisen et al., 2018; Adl et al., 2012).

Recommendations for identification

For effective identification of protists, it is recommended to:

1. Use morphological methods (light and electron microscopy, staining) for initial characterization.

2. Apply molecular methods (PCR, metabarcoding) for accurate taxonomic identification.

3. Use quantitative methods (MPN, qPCR, ddPCR) to assess abundance.

4. Combine data on morphology, genetics, and ecology for comprehensive characterization (Abraham et al., 2019; Geisen et al., 2018).

Protists are a polyphyletic group of eukaryotes

As we found out, protists do not form a monophyletic group. This is a polyphyletic assemblage of eukaryotic organisms that do not belong to animals, plants, or fungi (Adl et al., 2012; Ruppert et al., 2008). Modern eukaryotic phylogeny distinguishes several supergroups that include protists: Amoebozoa, Excavata, SAR (Stramenopiles, Alveolata, Rhizaria), Archaeplastida, and others (Adl et al., 2012; Geisen et al., 2018).

The term “protists” retains its significance as a convenient designation for organisms traditionally studied by protozoology and algology, but it does not reflect their evolutionary relationships (Ruppert et al., 2008; Hickman et al., 2004).

Enormous taxonomic and functional diversity

We have seen that soil protists are represented by diverse groups: amoebae (naked and testate), slime molds, flagellates (heterotrophic and phototrophic), ciliates (predatory, bacterivorous, detritivorous), cercozoans (testate amoebae, heliozoans), apicomplexans, and photosynthetic protists (diatoms, green algae, cyanobacteria) (Bonkowski et al., 2012; Geisen et al., 2018).

Molecular methods (18S rRNA metabarcoding) reveal a huge number of previously unknown protist lineages, indicating a significant underestimation of their diversity (Lentendu et al., 2022; Geisen et al., 2018).

Key role in the soil microbiome

We have shown that protists play a central role in the functioning of the soil microbiome. They participate in the bacterial loop, controlling bacterial numbers and releasing nutrients in a form available to plants (Clarholm, 1985; Bonkowski et al., 2012). Protists stimulate plant growth both directly (through the release of phytohormones) and indirectly (through improved nutrition and soil structure) (Bonkowski et al., 2012; Geisen et al., 2018).

Protists are capable of suppressing phytopathogens both directly (by consuming them) and indirectly (by stimulating the activity of antagonistic bacteria), which makes them promising biocontrol agents (Fujino et al., 2025). They can also be used as indicators of soil health due to their sensitivity to changes in conditions and short generation time (Geisen et al., 2018).

Parasitic protists of plants

We examined parasitic protists of plants. Plasmodiophora brassicae — the causative agent of clubroot of cabbage — belongs to the phytomyxea (Rhizaria, Cercozoa) (Webster & Weber, 2007; Adl et al., 2012). Spongospora subterranea — the causative agent of powdery scab of potato — is also a phytomyxean (Webster & Weber, 2007). Polymyxa spp. — vectors of dangerous plant viruses, including beet necrotic yellow vein virus (BNYVV) (Draycott, 2006).

We also discussed the differences between parasitic protists and fungi and oomycetes in cell wall structure, type of nutrition, life cycles, and genetic characteristics (Webster & Weber, 2007; Agrios, 2005; Alexopoulos, 1996).

Other ecological groups of protists

We examined other ecological groups of protists in agroecosystems: phototrophic (soil algae, cyanobacteria), saprotrophic (oomycetes, some flagellates), symbiotic (lichens, mixotrophs), parasites of animals (coccidia, piroplasms), and human parasites (Giardia, Entamoeba, Toxoplasma, Cryptosporidium, Acanthamoeba) (Geisen et al., 2018; Hickman et al., 2004; Ruppert et al., 2008; Weil, 2017).

We also discussed the role of free-living amoebae as reservoirs for pathogenic bacteria — the “Trojan horse” phenomenon of the microbial world (Bonkowski et al., 2012; Geisen et al., 2018).

Practical importance for agronomy

We have shown that knowledge about protists has direct practical importance for agronomy. Molecular methods (PCR, qPCR, ddPCR) make it possible to diagnose protist diseases at early stages and with high accuracy (Tso et al., 2021). Protists can be used as indicators of soil condition (Geisen et al., 2018; Bonkowski et al., 2012). Predatory protists are promising as biocontrol agents against phytopathogens (Fujino et al., 2025). Agrotechnologies (tillage, fertilizers, pesticides, crop rotation) have a significant impact on protist communities (Geisen et al., 2018; Weil, 2017). Protists are promising for bioremediation of contaminated soils (Geisen et al., 2018).

Methods of protist identification

We examined methods of protist identification: morphological (light and electron microscopy, special staining methods), cultural (isolation, cloning, MPN), molecular (PCR, 18S rRNA metabarcoding, qPCR, ddPCR) (Abraham et al., 2019; Lentendu et al., 2022; Tso et al., 2021). We also discussed the problems of identification: cryptic species, polymorphism, incompleteness of databases, primer bias, introns, and quantitative limitations (Geisen et al., 2018; Lentendu et al., 2022).

Description of taxonomic diversity

One of the main tasks remains the description of the taxonomic diversity of protists. As noted by Geisen et al. (2018), most protist species have not yet been described, and their bioindicator potential is unknown. Large-scale studies are needed to inventory protists in different types of soils and agroecosystems.

Study of functional role

The second important task is to study the functional role of protists in soil processes. As noted by Geisen et al. (2018), we still poorly understand what specific functions different groups of protists perform and how they interact with other components of soil biota.

Development of biocontrol methods

A promising direction is the development of biocontrol methods for phytopathogens using predatory protists. As noted by Fujino et al. (2025), further research is needed on screening protists effective against specific pathogens, developing methods for their mass cultivation, and conducting field trials.

Use of protists as bioindicators

Another promising direction is the use of protists as bioindicators of soil condition. As noted by Geisen et al. (2018), with the development of molecular methods and accumulation of data, protists may become an important tool for assessing soil health in agroecosystems.

Bioremediation

Protists are promising for bioremediation of contaminated soils due to their ability to degrade organic pollutants and accumulate heavy metals. As noted by Geisen et al. (2018), further research is needed on screening protists effective in degrading specific pollutants and studying the synergy of protists and bacteria in bioremediation.

Impact of climate change

An urgent task is to study the impact of climate change on protist communities. As noted by Geisen et al. (2018), rising temperatures, changing precipitation patterns, and other climatic factors can significantly affect the composition and functioning of protist communities, which in turn may affect the productivity of agroecosystems.

Integration of protistology into agricultural practice

Finally, an important task is the integration of knowledge about protists into agricultural practice. As noted by Geisen et al. (2018), it is necessary to develop practical recommendations for agronomists on managing protist communities to increase the productivity and sustainability of agroecosystems.