Nematodes: Phytoparasitic and Soil

1.1. Introduction

Nematodes, or roundworms, are one of the most numerous and ubiquitous groups of multicellular animals on Earth. They belong to the phylum Nematoda, which together with the closely related phylum Nematomorpha (horsehair worms) and the superphylum Panarthropoda (arthropods, onychophorans, and tardigrades) is part of the clade Ecdysozoa (moulting animals). Thus, nematodes are not arthropods and not “worms” in the everyday sense, but a separate, evolutionarily distinct phylum of animals possessing a unique set of morphological, physiological, and ecological features (Schierwater, 2022; Perry, 2013).

The name “nematode” comes from the Greek nema — “thread” and oides — “like,” which reflects their typical filamentous or spindle-shaped body (Reddy, 2019). In Russian-language literature they are also called “nematodes,” “roundworms,” or “nematodes-nematodes”; in English — “nematodes,” “roundworms,” “eelworms” (the latter for phytoparasitic forms) (Agrios, 2005).

1.2. Abundance and Biomass

Nematodes are probably the most numerous group of multicellular animals on the planet. The American nematologist N. A. Cobb back in 1915 figuratively remarked: “If all the matter in the universe except nematodes were removed, our world would still be vaguely recognizable — we would find its mountains, hills, valleys, rivers, lakes, and oceans represented by a film of nematodes” (cited in Schierwater, 2022; Reddy, 2019). Although this statement is hyperbole, it accurately conveys the scale of their distribution and abundance.

Modern estimates of nematode abundance are impressive. A single gram of soil may contain hundreds to thousands of individuals, and in the upper soil layer over one hectare — billions to trillions of nematodes (Weil, 2017). For example, Table 11.4 (Weil, 2017) indicates that nematode abundance in surface soil horizons can reach 10^5–107 individuals per m^{2, and biomass — 1–30 g/m}2 in live weight. By comparison, earthworm biomass under the same conditions may be 10–400 g/m^{2, and microarthropod biomass — 0.2–5 g/m}2. Thus, nematodes, while inferior in biomass to earthworms, significantly exceed most other groups of soil fauna in abundance.

The total number of described nematode species exceeds 28,000 (Schierwater, 2022), although some estimates suggest actual species diversity may range from 100,000 to 1 million species or more (Weil, 2017; Schierwater, 2022). For example, a single gram of soil may contain several thousand species of bacteria and hundreds of nematode species (Weil, 2017). Dozens of new species are described annually, especially in tropical and poorly studied regions.

The global biomass of nematodes is enormous. It is estimated that the total biomass of nematodes in the Earth’s soils is comparable to or even exceeds the biomass of all terrestrial arthropods. In marine bottom sediments, nematodes also dominate among meiobenthos, accounting for up to 90% of the abundance of all multicellular animals (Schierwater, 2022).

1.3. Distribution

Nematodes are distributed everywhere: from Arctic ice to tropical forests, from deep-ocean trenches to high mountains, from freshwater bodies to saline soils and hot springs (Reddy, 2019; Schierwater, 2022). They occur in soil, water, plants, and the bodies of animals and humans. There is perhaps no habitat on Earth where nematodes have not been found, except possibly some extreme environments, but even there they are represented by specialized forms.

In soil, nematodes inhabit pore spaces filled with water or air. They can actively move in water films covering soil particles or in capillary pores. Their vertical distribution depends on moisture, temperature, presence of organic matter, and plant roots. The highest density is usually observed in the upper 15–30 cm of soil, but some species penetrate to depths of several meters (Agrios, 2005; Weil, 2017).

In agricultural soils, nematodes are also abundant and diverse. They can be free-living (bacteriophagous, fungivorous, predatory) or parasitic on plants. It is estimated that annual global crop losses from phytoparasitic nematodes amount to about 11% for major food crops (Agrios, 2005) and more than 173 billion USD per year (Caliskan et al., 2023). This makes nematodes one of the most economically significant groups of organisms in the agrosphere.

1.4. Role in Soil Ecosystems

Nematodes play a key role in soil food webs and ecosystem functioning. They occupy several trophic levels: from primary consumers (herbivorous forms) to second- and third-order consumers (predators, omnivores). By feeding type, soil nematodes are divided into several functional groups: bacterivores (feed on bacteria), fungivores (feed on fungi), herbivores (root parasites), predators (eat other nematodes, protozoa, small invertebrates), omnivores, and parasites of invertebrates (Weil, 2017; Huang, 2012).

Through food webs, nematodes participate in the decomposition of organic matter and the cycling of biogenic elements (nitrogen, phosphorus, carbon). By eating bacteria and fungi, they release excess nitrogen in the form of ammonium, which becomes available to plants. It is believed that bacterivorous nematodes can provide up to 30–40% of mineralized nitrogen in some ecosystems (Weil, 2017). In addition, nematodes serve as food for larger soil organisms (microarthropods, enchytraeids, earthworms), thereby transferring energy to higher trophic levels.

Nematodes are also indicators of soil condition and anthropogenic load. Their species composition, abundance, and ratio of trophic groups are sensitive to changes in pH, moisture, temperature, contamination with heavy metals, pesticides, organic fertilizers, and other factors. Based on nematode community indices (e.g., maturity index, cp-classes), methods for soil biodiagnostics have been developed (Huang, 2012; Weil, 2017).

Table 11.4 (Weil, 2017) provides data on nematode abundance and biomass in soil, which emphasize their significant contribution to the total biomass of soil organisms, although it is inferior to bacteria and fungi.

1.5. Role in Agriculture

In agrocoenoses, nematodes can cause both harm and benefit.

Harm. Phytoparasitic nematodes are one of the main groups of agricultural crop pests. They affect the root system, underground organs (tubers, bulbs, root crops), and some — aboveground plant parts. Among the most harmful groups: cyst nematodes (Heterodera, Globodera), root-knot nematodes (Meloidogyne), stem nematodes (Ditylenchus dipsaci), leaf nematodes (Aphelenchoides), root nematodes (Pratylenchus, Radopholus), and virus-vector nematodes (Xiphinema, Longidorus, Trichodorus). Crop losses from them are estimated at billions of dollars annually (Agrios, 2005; Caliskan et al., 2023). For example, the potato cyst nematodes Globodera rostochiensis and G. pallida alone cause losses of up to 9% of world potato production, and in some regions — up to 50–80% (Caliskan et al., 2023). Root-knot nematodes Meloidogyne spp. affect more than 3,000 plant species, including tomatoes, cucumbers, peppers, potatoes, and can reduce yields by 30–70% (Reddy, 2019).

Benefit. Entomopathogenic nematodes (EPNs) from the genera Steinernema and Heterorhabditis are used for biological control of insect pests. They are symbiotic with bacteria Xenorhabdus and Photorhabdus, which kill insects within 24–48 hours. EPNs are effective against soil pests (beetle larvae, cutworms, wireworms, weevils) and cryptic forms (e.g., root pests) (Vashisth, 2013; Grewal, 1999). They are safe for non-target organisms, compatible with many pesticides, and can be used in integrated plant protection systems. However, their use is limited by sensitivity to UV radiation, low humidity, high temperatures, and production cost (Vashisth, 2013).

Free-living soil nematodes, in particular Caenorhabditis elegans, serve as model objects in biology, genetics, toxicology, and ecotoxicology. C. elegans was the first multicellular organism with a fully sequenced genome, making it a key tool for studying development, neuroscience, aging, and interactions with pathogens (Schierwater, 2022; Reddy, 2019).

2.1. Introduction

The systematics and phylogeny of nematodes are among the most complex and dynamically developing fields of zoology. Traditionally, nematode classification was based on morphological characters: structure of the cuticle, head capsule, stylet, esophagus, tail, and reproductive system. However, the morphological simplicity and uniformity of the basic body plan, as well as the high plasticity of many characters, led to numerous taxonomic problems: paraphyly, polyphyly, and frequent revisions of the system (Schierwater, 2022; De Ley & Blaxter, 2002). Since the late 20th century, molecular phylogenetic methods, primarily analysis of ribosomal RNA gene sequences (18S, 28S, ITS) and mitochondrial DNA (COI), have radically changed ideas about relationships within the phylum Nematoda. The modern system of nematodes is the result of a synthesis of morphological, molecular, and ecological data, although many questions are still far from final resolution (Perry, 2013; Schierwater, 2022).

2.2. Position of Nematoda in the Animal System

As already noted in the first chapter, Nematoda is a separate phylum within the clade Ecdysozoa (moulting animals). Phylogenetic studies based on analysis of 18S and 28S rRNA genes, as well as whole-genome data, confirm that the closest relatives of nematodes are Nematomorpha (horsehair worms), and together they form a sister group to Panarthropoda (Arthropoda, Onychophora, Tardigrada) (Schierwater, 2022; Blaxter et al., 1998). Some molecular analyses place Nematoda + Nematomorpha closer to Tardigrada, but these results are often explained by artifacts associated with substitution saturation and homoplasy (Campbell et al., 2011).

The systematic position of Nematoda within Ecdysozoa can be represented as follows:

Ecdysozoa

• Nematoida (Nematoda + Nematomorpha)
• Panarthropoda (Arthropoda, Onychophora, Tardigrada)
• Scalidophora (Priapulida, Kinorhyncha, Loricifera)

This scheme reflects modern molecular phylogenetic data, although the position of some groups (e.g., Loricifera) remains debated (Schierwater, 2022; Giribet & Edgecombe, 2017).

Apomorphies uniting Nematoda with other Ecdysozoa include the presence of a cuticle consisting of collagen and chitin (in nematodes, chitin is present only in the egg shell, not in the cuticle) and periodic moulting (ecdysis) regulated by ecdysteroid hormones. Unique apomorphies of Nematoda: absence of ciliated locomotor cells, presence of longitudinal musculature of four bands, pseudocoelom, a peculiar nervous system with a ventral nerve cord and a nerve ring around the pharynx, and amoeboid sperm using MSP (major sperm protein) instead of actin for movement (Schierwater, 2022).

2.3. Modern Phylogeny and System of Nematoda

2.4. Modern Molecular Data and Revision of the System

Molecular phylogenetic studies, beginning with analysis of the 18S rRNA gene (Blaxter et al., 1998), and then expanded using 28S, ITS, mitochondrial genes (COI, cytb), and whole-genome data, have led to a substantial revision of the Nematoda system. Main changes:

1. Rejection of old groupings. The traditional classes Secernentea and Adenophora (or Phasmidia and Aphasmidia) were recognized as paraphyletic. The modern system uses clades designated by Roman numerals (I–V) or names (Enoplia, Dorylaimia, Chromadoria) (De Ley & Blaxter, 2002; Schierwater, 2022).

2. Polyphyly of Tylenchida. rRNA analysis showed that Tylenchida (in the traditional scope) is not a monophyletic group; phytoparasitism arose independently in several lineages (Tylenchina, Aphelenchina, Dorylaimida) (Blaxter et al., 1998; Schierwater, 2022).

3. Relationships within Chromadoria. It has been established that Rhabditida (in the broad sense) includes Tylenchina, Aphelenchina, Rhabditina, Spirurina, and other groups. This is confirmed by both nuclear and mitochondrial genes (Kiontke et al., 2007; Schierwater, 2022).

4. Phylogeny of Enoplia and Dorylaimia. Enoplia is a basal clade, but its internal phylogeny remains poorly resolved. Dorylaimia, including Longidoridae, demonstrates close relationship with Mermithida and Trichinellida, confirming the independent origin of parasitism in this lineage (Perry, 2013).

5. Use of whole-genome data. Sequencing of genomes of more than 100 nematode species (including C. elegans, Pristionchus pacificus, Bursaphelenchus xylophilus, Meloidogyne spp., Globodera spp.) has refined phylogenetic relationships and identified genes associated with parasitism (Schierwater, 2022; International Helminth Genomes Consortium, 2019).

Despite progress, many questions remain open: phylogeny of Enoplia, position of some families (e.g., Trichodoridae), boundaries of genera and species, and mechanisms of the evolution of parasitism.

2.5. The Problem of Cryptic Species and Species Complexes

One of the key problems in nematode systematics is the presence of cryptic species (morphologically indistinguishable but genetically isolated) and species complexes. This is especially characteristic of phytoparasitic and parasitic forms, where morphological characters are often limited and subject to plasticity.

Examples of species complexes:

• Meloidogyne spp. — root-knot nematodes. The complex M. incognita, M. arenaria, M. javanica, M. hapla, etc. includes many cryptic species differing in biochemical (esterase phenotypes), molecular (ITS, SCAR markers), and biological (host range, virulence) characters (Caliskan et al., 2023; Perry, 2013). For example, M. mayaguensis was synonymized with M. enterolobii, but for a long time was considered a separate species, causing confusion in quarantine measures (Perry, 2013).
• Pratylenchus spp. — lesion nematodes. More than 70 described species, many of which overlap morphologically. Identification uses molecular markers (ITS rDNA, D2-D3 28S) and isoenzyme analysis (Caliskan et al., 2023; Perry, 2013).
• Globodera rostochiensis and G. pallida — potato cyst nematodes. Although they clearly differ in female color and some morphometric characters, within each species there are pathotypes and virulent groups requiring molecular diagnostics (Perry, 2013).
• Bursaphelenchus xylophilus and B. mucronatus — pine nematodes. Morphologically close but differ in pathogenicity and molecular markers (ITS, IGS, satellite DNA) (Perry, 2013).
• Radopholus similis and R. citrophilus — burrowing nematodes. For a long time considered different species (banana and citrus races), then synonymized, but the question remains debatable (Perry, 2013).

The problem of cryptic species has direct practical importance for quarantine and plant protection: incorrect identification can lead to ineffective control measures or unjustified trade restrictions. Therefore, modern nematode diagnostics necessarily includes molecular methods, often in combination with morphology (integrative taxonomy) (Perry, 2013; Caliskan et al., 2023).

3.1. Introduction

The morphology and anatomy of nematodes represent a unique combination of a conservative basic body plan and significant variability of individual structures, which is of primary importance for systematics, diagnostics, and understanding the biology of these animals. Despite enormous species diversity and a wide range of ecological niches, all nematodes share a common body plan: the body is a “tube within a tube” — the inner tube (digestive system) is surrounded by the outer tube (body wall), and between them is a fluid-filled primary body cavity (pseudocoelom) that functions as a hydrostatic skeleton (Schierwater, 2022; Decraemer & Hunt, 2013). It is this simplicity and universality that allow nematodes to successfully exist in a variety of environments, and their morphological characters reliably identify taxa.

In this chapter we will consider the main organ systems of nematodes, their structure and functions, as well as characters used in systematics and diagnostics. Particular attention will be paid to structures important for identification of phytoparasitic and soil forms: cuticle, head capsule, stylet, esophagus, tail, and reproductive system.

3.2. General Body Plan

The body of nematodes is usually elongated, spindle-shaped or filamentous, round in cross-section, with bilateral symmetry. Body length varies from 0.2 mm (e.g., Paratylenchus) to 12 mm or more (e.g., Paralongidorus epimikis), but in most species is about 1 mm (Reddy, 2019; Decraemer & Hunt, 2013). The body is covered by a cuticle, beneath which is the hypodermis (epidermis), and then longitudinal musculature. Internal organs are located in the pseudocoelom.

Anatomically, the body is divided into four longitudinal sectors: dorsal, ventral, and two lateral. Ventrally located are the excretory pore, vulva, and anus (or cloaca in males); laterally — amphids, deirids, and phasmids (Decraemer & Hunt, 2013). The anterior end bears the mouth opening, surrounded by lips and sensilla.

3.3. Body Wall

3.4. Body Cavity (Pseudocoelom)

The pseudocoelom is a fluid-filled cavity not lined by epithelium. It is surrounded by muscles and a basal lamina, contains pseudocoelomic cells (coelomocytes) performing storage, excretory, and transport functions. The fluid of the pseudocoelom creates turgor pressure, maintaining body shape and participating in the hydrostatic skeleton (Reddy, 2019; Decraemer & Hunt, 2013).

3.5. Digestive System

The digestive system of nematodes is a through tube consisting of three sections: stomodeum (foregut), mesenteron (midgut), and proctodeum (hindgut). The stomodeum and proctodeum are of ectodermal origin and lined with cuticle; the mesenteron is endodermal (Schierwater, 2022; Reddy, 2019).

3.6. Excretory System

The excretory system of nematodes can be represented by:

• Glandular type — one large cell (renette) with a duct opening to the outside (e.g., in Longidoridae) (Decraemer & Hunt, 2013);
• Tubular type — an H-shaped or U-shaped system of canals located in the lateral chords, with an excretory pore (in most Chromadoria) (Reddy, 2019);
• Asymmetric type — one lateral canal (in Tylenchida) (Reddy, 2019).

Function — osmoregulation and excretion of metabolic products.

3.7. Nervous System and Sense Organs

The nervous system of nematodes consists of a nerve ring (circumpharyngeal) surrounding the pharynx and longitudinal nerve cords: ventral (most developed), dorsal, and two lateral. Nerves extend from the ring to sensilla of the anterior end. Ganglia are located in the head end and in the anal region (Schierwater, 2022; Reddy, 2019).

Sense organs are represented by sensilla:

• Amphids — paired chemoreceptors located laterally on the head; their shape (pocket, spiral, loop) is important for systematics (Decraemer & Hunt, 2013).
• Phasmids — paired chemoreceptors in the tail region (in Tylenchida and Rhabditida).
• Papillae and setae — mechano- and chemoreceptors on lips and body.
• Deirids — mechanoreceptors in the neck region.

Some marine nematodes have eyespots (ocelli), but phytoparasitic forms lack them (Schierwater, 2022).

3.8. Reproductive System and Sexual Dimorphism

3.9. Characters Used in Systematics and Diagnostics

For identification of nematodes, the following morphological characters are used:

• Head capsule: shape, presence or absence of ring thickenings, cuticle sculpture.
• Stylet: length, shape of knobs, presence of guiding ring (in Dorylaimida).
• Esophagus: type (tylenchoid, dorylaimoid, rhabditoid), number of bulbs, location of esophageal glands.
• Tail: shape (conical, blunt, pointed), presence of mucro, hyaline zone, phasmids.
• Reproductive system: number of ovaries, vulva position (V%), presence of spermatheca, shape of spicules and gubernaculum, number of precloacal papillae.
• Cuticle: annulation, lateral fields, wings, perineal pattern (in Meloidogyne), scars on cysts (in Heterodera, Globodera).

These characters, in combination with morphometric indices (a, b, c, V, c'), allow species identification (Reddy, 2019; Decraemer & Hunt, 2013).

4.1. Introduction

Phytoparasitic nematodes are a vast and economically significant group of organisms parasitizing higher plants. They belong to different taxonomic groups within the phylum Nematoda but are united by the ability to use plants as a source of food and habitat. It is estimated that annual global crop losses from phytoparasitic nematodes amount to about 11% for major food crops and more than 173 billion USD per year (Agrios, 2005; Caliskan et al., 2023). In this chapter we will consider the main groups of phytoparasitic nematodes, their biology, symptoms of diseases they cause, and practical significance.

Phytoparasitic nematodes are divided into several ecological groups depending on the mode of parasitism:

• Ectoparasites — feed from outside the root without penetrating tissues (e.g., Xiphinema, Longidorus, Trichodorus).
• Endoparasites — penetrate plant tissues; among them, migratory (e.g., Pratylenchus, Radopholus) and sedentary (e.g., Meloidogyne, Heterodera, Globodera) forms are distinguished.
• Semi-endoparasites — anterior end of the body is embedded in tissue, posterior remains outside (e.g., Tylenchulus semipenetrans, Rotylenchulus).
• Aboveground parasites — affect stems, leaves, flowers, seeds (e.g., Ditylenchus dipsaci, Aphelenchoides spp., Anguina spp.).

Most phytoparasitic nematodes belong to the order Tylenchida (class Chromadorea), except for longidorid and needle nematodes (family Longidoridae), which belong to the order Dorylaimida (class Enoplea) (Perry, 2013; Decraemer & Hunt, 2013).

4.2. Cyst Nematodes: Heterodera and Globodera

Cyst nematodes are one of the most harmful groups of phytoparasitic nematodes. They belong to the family Heteroderidae (Tylenchida). The genus Heterodera includes about 80 species, the genus Globodera — 12 species (Perry, 2013). The greatest economic importance is attached to Heterodera schachtii (sugar beet cyst nematode), H. glycines (soybean cyst nematode), H. avenae (oat cyst nematode), Globodera rostochiensis and G. pallida (potato cyst nematodes, or golden and pale potato nematodes) (Agrios, 2005; Caliskan et al., 2023).

4.3. Root-Knot Nematodes: Meloidogyne

Root-knot nematodes (Meloidogyne spp.) are the most economically significant group of phytoparasitic nematodes in the world. They affect more than 3,000 plant species, including almost all vegetable, industrial, and ornamental crops. The genus includes about 100 species, of which the most harmful are M. incognita, M. javanica, M. arenaria, M. hapla, M. chitwoodi, M. fallax (Caliskan et al., 2023; Agrios, 2005).

4.4. Stem and Bulb Nematodes: Ditylenchus dipsaci

Ditylenchus dipsaci — stem and bulb nematode, affecting about 500 plant species, including onion, garlic, narcissus, tulip, clover, alfalfa, potato, beet, strawberry. It belongs to the family Anguinidae (Tylenchida). The nematode is an endoparasite of aboveground organs: stems, leaves, flowers, bulbs, tubers (Agrios, 2005; Perry, 2013).

4.5. Leaf Nematodes: Aphelenchoides

Aphelenchoides spp. — leaf nematodes parasitizing aboveground plant organs. The most important species: A. ritzemabosi (chrysanthemum nematode), A. fragariae (strawberry nematode), A. besseyi (white-tip nematode of rice). They belong to the family Aphelenchoididae (Tylenchida). They affect chrysanthemums, strawberries, begonias, violets, ferns, rice, coconut palm (Agrios, 2005; Perry, 2013).

4.6. Root Nematodes: Pratylenchus (Lesion), Radopholus (Burrowing)

4.7. Ectoparasitic Nematodes — Virus Vectors: Xiphinema and Longidorus

Xiphinema (dagger nematodes) and Longidorus (needle nematodes) are ectoparasitic nematodes of the family Longidoridae (Dorylaimida). They possess a long hollow stylet (odontostyle) and feed on root cells, causing necroses and galls. But their main importance is transmission of nepoviruses (Nepoviruses), such as grapevine fanleaf virus (GFLV), tobacco ringspot virus (TRSV), tomato black ring virus (ToRSV), etc. (Perry, 2013; Decraemer & Geraert, 2013).

4.8. Other Important Phytoparasitic Nematodes

5.1. Introduction

Accurate identification of phytoparasitic nematodes is a necessary basis for phytosanitary diagnostics, quarantine measures, harmfulness forecasting, and selection of effective plant protection measures. Errors in species determination can lead to unjustified trade restrictions, ineffective use of nematicides, spread of dangerous quarantine objects, and, as a consequence, significant economic losses (Perry, 2013; Hockland et al., 2013). Unlike many other groups of organisms, identification of nematodes is complicated by their small size, morphological conservatism, wide distribution of cryptic species and species complexes, and frequent presence of mixed populations in soil and plant tissues (Caliskan et al., 2023; Perry, 2013).

Modern nematode diagnostics is based on an integrative approach combining classical morphological and morphometric methods with biochemical and molecular-genetic methods. Such an approach allows reliable distinction of closely related species and detection of intraspecific variability, pathotypes, and virulent groups, which is especially important for quarantine species (Perry, 2013; EPPO, 2013).

This chapter discusses the main methods for identification of phytoparasitic nematodes: extraction from soil and plant tissues, morphological and morphometric studies, biochemical methods, molecular approaches, and problems and prospects of diagnostics.

5.2. Extraction of Nematodes from Soil and Plant Tissues

The first stage of identification is extraction of nematodes from the sample. The reliability of subsequent determination largely depends on the efficiency and purity of extraction. For extraction of phytoparasitic nematodes from soil and plants, standardized methods described in EPPO guidelines (EPPO, 2013) and other sources (Huang, 2012; Perry, 2013) have been developed.

5.3. Morphological and Morphometric Methods

5.4. Biochemical Methods

Biochemical methods are based on analysis of proteins and enzymes of nematodes. They allow distinction of closely related species and pathotypes, especially where morphological characters overlap.

• Isoenzyme analysis — electrophoresis in starch or polyacrylamide gel. Esterases, malate dehydrogenase, phosphoglucomutase are most often used. For root-knot nematodes (Meloidogyne), esterase phenotypes have been developed: A1, A2, A3 for M. arenaria; Ch1 for M. chitwoodi; F3 for M. fallax; H1 for M. hapla; I1, I2, I3 for M. incognita; J3 for M. javanica (Caliskan et al., 2023; Perry, 2013).
• Isoelectric focusing (IEF) — separation of proteins by isoelectric point. Used for diagnostics of Globodera rostochiensis and G. pallida (Perry, 2013).
• Immunological methods — monoclonal antibodies, ELISA. Used for detection of nematodes in soil and plants, but limited due to antigen variability (Perry, 2013).

Biochemical methods require living nematodes, which limits their application to fixed samples. In addition, they are sensitive to cultivation conditions and physiological state of nematodes (Perry, 2013).

5.5. Molecular Methods

Molecular-genetic methods have revolutionized nematode diagnostics. They allow identification of species and intraspecific groups regardless of developmental stage, sex, and morphological variability, as well as detection of nematodes in mixed populations and in soil without preliminary extraction (Perry, 2013; Caliskan et al., 2023).

5.6. Problems of Identification

Despite progress, identification of phytoparasitic nematodes remains a difficult task. Main problems:

• Cryptic species and species complexes. Many species are morphologically indistinguishable but genetically isolated. Examples: Meloidogyne spp., Pratylenchus spp., Globodera spp., Radopholus similis / R. citrophilus, Bursaphelenchus xylophilus / B. mucronatus (Perry, 2013; Caliskan et al., 2023).
• Intraspecific variability. Morphometric characters can vary significantly depending on host plant, temperature, and developmental stage. For example, in Pratylenchus tail shape and vulva position may overlap between species (Caliskan et al., 2023).
• Mixed populations. A single sample may contain several species, complicating morphological identification. Molecular methods allow detection of each species separately (Perry, 2013).
• Lack of reference sequences. For many species, especially poorly studied ones, reliable molecular markers are absent, limiting diagnostics (Schierwater, 2022).
• Quarantine species. For quarantine nematodes (Bursaphelenchus xylophilus, Meloidogyne chitwoodi, M. fallax, Globodera rostochiensis, G. pallida, Xiphinema americanum sensu lato), standardized EPPO protocols have been developed, including morphological and molecular methods (EPPO, 2013; Hockland et al., 2013).
• Polymorphism and hybridization. In some species (e.g., Meloidogyne), hybridization and polyploidy are possible, complicating interpretation of molecular data (Caliskan et al., 2023).

5.7. Quarantine Species and Their Diagnostics

For quarantine nematodes, special diagnostic protocols approved by EPPO and other international organizations have been developed. They include:

• Sampling — soil, plants, wood, seeds.
• Nematode extraction — standardized methods (EPPO, 2013).
• Morphological identification — using light and electron microscopy, morphometrics.
• Molecular identification — PCR with species-specific primers, PCR-RFLP, qPCR, LAMP, sequencing (EPPO, 2013; Hockland et al., 2013).
• Confirmation — by independent methods, preferably in an accredited laboratory.

The most important quarantine species include:

• Bursaphelenchus xylophilus (pine nematode) — quarantine object in the EU, Russia, China, Japan, etc. (Hockland et al., 2013).
• Globodera rostochiensis, G. pallida (potato cyst nematodes) — quarantine objects in many countries (Perry, 2013).
• Meloidogyne chitwoodi, M. fallax, M. enterolobii — quarantine species in the EU and other regions (Caliskan et al., 2023; Hockland et al., 2013).
• Radopholus similis / R. citrophilus — quarantine species for citrus and bananas (Hockland et al., 2013).
• Xiphinema americanum sensu lato, X. index, X. californicum — vectors of nepoviruses, quarantine objects (Hockland et al., 2013).

6.1. Introduction

Free-living soil nematodes are a vast and ecologically important group of nematodes that do not parasitize plants or animals but inhabit soil, feeding on bacteria, fungi, protozoa, other nematodes, or organic remains. They occur in almost all soils of the world, from Arctic tundra to tropical forests, and play a key role in soil food webs, decomposition of organic matter, and cycling of biogenic elements (Weil, 2017; Huang, 2012). Free-living nematodes constitute a significant part of soil microfauna: their abundance in upper soil horizons can reach 10^5–107 individuals per m^{2, and biomass — 1–30 g/m}2 in live weight (Weil, 2017, Table 11.4). Unlike phytoparasitic forms, they do not cause direct harm to agricultural crops, but their presence and species composition serve as an important indicator of soil condition and anthropogenic load (Huang, 2012; Weil, 2017).

In this chapter we will consider the diversity of free-living soil nematodes, their trophic groups, role in the soil food web, use as bioindicators, and the importance of Caenorhabditis elegans as a model object.

6.2. Diversity and Classification of Free-Living Soil Nematodes

Free-living soil nematodes do not form a single taxonomic group; they occur in several large clades of the phylum Nematoda. According to the modern system (Schierwater, 2022; De Ley & Blaxter, 2002), among soil free-living forms the most common are representatives of:

1) Enoplia (Clade I) — mainly marine and soil forms, such as Enoplus, Tobrilus, Pontonema. They are less common in soils but can be indicators of undisturbed habitats.

2) Dorylaimia (Clade II) — include free-living soil nematodes (Dorylaimida), as well as predatory Mononchida. Dorylaimida includes large omnivorous and predatory forms (Dorylaimus, Eudorylaimus, Aporcelaimellus), as well as ectoparasitic Longidoridae (discussed in Chapter 4).

3) Chromadoria (Clades III–V) — the most numerous group, including most free-living soil nematodes. Among them:

• Rhabditida — bacterivorous forms (Caenorhabditis, Acrobeloides, Cephalobus, Panagrolaimus), as well as entomopathogenic Steinernematidae and Heterorhabditidae (discussed in Chapter 7).
• Tylenchida (Tylenchina) — mainly phytoparasitic forms, but among them there are also free-living fungivorous species (Filenchus, Tylenchus).
• Aphelenchida (Aphelenchina) — fungivorous forms (Aphelenchus, Aphelenchoides), some of which can also feed on plant cells.
• Plectida, Chromadorida, Desmodorida, Monhysterida — free-living marine and soil forms occurring in moist soils.

In soils of temperate and tropical latitudes, the most common and numerous are representatives of the genera Acrobeloides, Cephalobus, Panagrolaimus, Rhabditis, Plectus, Wilsonema, Tylenchus, Filenchus, Aphelenchus, Dorylaimus, Eudorylaimus, Mononchus, etc. (Huang, 2012; Weil, 2017).

6.3. Role in the Soil Food Web

Free-living soil nematodes occupy several trophic levels and participate in key ecosystem processes. In the soil food web they function as:

• Primary consumers (bacterivores and fungivores) — feed on bacteria and fungi, regulating their abundance and activity.
• Secondary consumers (predators and omnivores) — eat other nematodes, protozoa, rotifers, small invertebrates.
• Decomposers — some species consume organic remains, promoting their decomposition.

Through food interactions, nematodes accelerate mineralization of organic matter and release of biogenic elements. For example, bacterivorous nematodes, by eating bacteria, release excess nitrogen in the form of ammonium, which becomes available to plants. It is believed that this process can provide up to 30–40% of mineralized nitrogen in some ecosystems (Weil, 2017). In addition, nematodes serve as food for larger soil organisms (microarthropods, enchytraeids, earthworms), transferring energy to higher trophic levels.

Table 11.4 (Weil, 2017) shows that nematodes are inferior to bacteria and fungi in biomass, but their contribution to soil metabolic activity is significant, especially in the rhizosphere. In the rhizosphere, where bacterial concentration is high, bacterivorous nematodes can reach high abundance and actively influence nitrogen cycling (Weil, 2017; Huang, 2012).

6.4. Trophic Groups of Soil Nematodes

By feeding type, free-living soil nematodes are divided into several functional groups (Huang, 2012; Weil, 2017; Yeates et al., 1993):

1. Bacterivores (bacteriophagous) — feed on bacteria. These include many Rhabditida (Caenorhabditis, Acrobeloides, Cephalobus, Panagrolaimus), Plectida (Plectus), and some Dorylaimida. They dominate in soils with high organic matter content and active bacterial biomass.

2. Fungivores (mycophagous) — feed on fungal mycelium. Represented by Tylenchida (Filenchus, Tylenchus), Aphelenchida (Aphelenchus, Aphelenchoides), and some Dorylaimida. They are common in forest litter and soils with developed fungal biota.

3. Herbivores (phytophagous) — parasitize plant roots (discussed in Chapter 4). In the context of free-living forms they are usually not considered, but some species can feed on root hairs without causing significant harm.

4. Predators — eat other nematodes, protozoa, rotifers, small invertebrates. These include Mononchida (Mononchus, Prionchulus), some Dorylaimida (Dorylaimus, Actinolaimus), and also some Rhabditida (Butlerius, Diplogaster). Predatory nematodes regulate the abundance of phytoparasitic and bacterivorous nematodes, playing the role of “top-down” regulators in the soil food web (Huang, 2012; Reddy, 2019).

5. Omnivores — feed on diverse food: fungi, bacteria, protozoa, other nematodes. Typical representatives — large Dorylaimida (Eudorylaimus, Aporcelaimellus). They occupy an intermediate position between predators and mycetophages (Huang, 2012; Yeates et al., 1993).

6. Parasites of invertebrates — some nematodes free-living in soil can parasitize insects, spiders, mollusks. They will be considered in Chapter 7 (entomopathogenic nematodes).

The ratio of trophic groups in a nematode community reflects the state of the soil ecosystem. For example, dominance of bacterivores indicates active decomposition of easily assimilable organic matter, while predominance of fungivores indicates the fungal decomposition pathway, characteristic of forest soils or soils with high lignin content (Weil, 2017; Huang, 2012).

6.5. cp-Classes and Maturity Index

To assess soil condition and the degree of anthropogenic load based on nematode communities, special indices have been developed. One of the most widely used is the maturity index (MI), proposed by Bongers (1990). It is based on the classification of nematodes by cp-classes (colonizer-persister scale), reflecting their life strategies and sensitivity to disturbances:

• cp-1 — rapidly reproducing, stress-tolerant species, pioneers of disturbed habitats (e.g., Rhabditis, Panagrolaimus, Acrobeloides).
• cp-2 — species with a short life cycle but less stress-tolerant (e.g., Cephalobus, Plectus).
• cp-3 — species with an average life cycle, moderately sensitive to disturbances (e.g., Tylenchus, Aphelenchus).
• cp-4 — long-lived, stress-sensitive species (e.g., Dorylaimus, Eudorylaimus).
• cp-5 — long-lived, very sensitive to disturbances species (e.g., Mononchus, Prionchulus).

Maturity index is calculated as the weighted average of cp-classes of all individuals in the community. High MI values indicate a stable, undisturbed ecosystem; low values indicate a disturbed one (Bongers, 1990; Huang, 2012; Weil, 2017).

Additionally used:

• Plant Parasite Index (PPI) — to assess the proportion of phytoparasitic nematodes.
• Enrichment Index (EI) — reflects availability of organic matter.
• Structure Index (SI) — reflects complexity of the food web.
• Channel Index (CI) — indicates predominance of bacterial or fungal decomposition pathway.

These indices are widely used in ecological monitoring, soil quality assessment, evaluation of the impact of agricultural practices (tillage, fertilization, pesticides), and restoration of disturbed lands (Huang, 2012; Weil, 2017).

6.6. Caenorhabditis elegans as a Model Object

Caenorhabditis elegans is a free-living bacterivorous nematode inhabiting soil and compost. It has become one of the most important model organisms in biology, medicine, and toxicology. C. elegans was the first multicellular organism with a fully sequenced genome (1998), making it a key tool for studying development, neuroscience, aging, cell biology, genetics, and interactions with pathogens (Schierwater, 2022; Reddy, 2019).

Main advantages of C. elegans as a model:

• Transparent body, allowing observation of cell development in real time.
• Short life cycle (3 days at 20 °C).
• Large progeny (up to 300 eggs per hermaphrodite).
• Ease of cultivation on agar with E. coli bacteria.
• Presence of hermaphrodites and males, facilitating genetic experiments.
• Fully described cell lineage (fate of each cell).
• Small genome size (_{100 Mb) and} 20,000 genes.
• Possibility of freezing and storing strains.

C. elegans is used to study:

• Apoptosis and cell death.
• RNA interference (RNAi).
• Neural networks and behavior.
• Aging and longevity.
• Toxicity of nanoparticles, heavy metals, pesticides.
• Host–pathogen interactions (e.g., with Pseudomonas aeruginosa, Salmonella).
• Evolutionary biology and ecology.

In the context of soil ecology, C. elegans serves as a model for understanding behavior, chemotaxis, stress response, and interaction with microorganisms. Transgenic strains of C. elegans with reporter genes (GFP, luciferase) are used for biotesting of soil and water pollution (Reddy, 2019; Schierwater, 2022).

6.7. Indicators of Soil Condition and Anthropogenic Load

Free-living soil nematodes are widely used as bioindicators due to their sensitivity to environmental changes and ease of sampling. Their communities respond to:

• Soil tillage — intensive ploughing reduces nematode abundance and diversity, especially cp-4 and cp-5 species, and increases the proportion of cp-1 and cp-2 (Weil, 2017).
• Fertilization — organic fertilizers stimulate bacterivores and increase total nematode abundance; mineral fertilizers can reduce diversity.
• Pesticides — nematicides and insecticides can sharply reduce nematode abundance, but some species are resistant.
• Heavy metal contamination — leads to reduced diversity and changes in the ratio of trophic groups.
• pH change — soil acidity affects community composition; for example, fungivores dominate in acidic soils.
• Moisture and temperature — seasonal fluctuations in abundance and composition.
• Vegetation cover — rhizosphere stimulates development of bacterivores and phytoparasites.

To assess soil condition, indices (MI, PPI, EI, SI, CI) are calculated, and the trophic structure of the community is analyzed. For example, a high MI value (>3) indicates a stable, undisturbed ecosystem; a low one (<2) — a disturbed one (Huang, 2012; Weil, 2017).

Modern molecular methods (qPCR, metabarcoding) allow more accurate and rapid assessment of nematode diversity and abundance in soil, opening new opportunities for monitoring and biodiagnostics (Campos-Herrera et al., 2015; Schierwater, 2022).

6.8. Methods for Studying Free-Living Soil Nematodes

For studying free-living nematodes, the same extraction methods are used as for phytoparasitic ones (Chapter 5): Baermann method, flotation, centrifugal flotation, elutriation (EPPO, 2013; Huang, 2012). After extraction, nematodes are fixed, slides are prepared, and morphological identification is carried out using light and electron microscopy. For quantitative counting, counting chambers or video-capture microscopy are used.

Molecular methods (PCR, qPCR, sequencing) allow identification of species and assessment of abundance without microscopy. For example, qPCR with species-specific primers is used for quantitative determination of Acrobeloides group, Cephalobus, Plectus, and others (Campos-Herrera et al., 2015). Metabarcoding by the 18S or COI gene makes it possible to characterize the entire nematode community in a sample (Schierwater, 2022; Weil, 2017).

For ecological studies, laboratory cultures of nematodes (e.g., C. elegans, Acrobeloides) are also important, allowing study of behavior, physiology, interactions with microorganisms, and toxicology (Reddy, 2019; Schierwater, 2022).

7.1. Introduction

Entomopathogenic nematodes (EPNs) are a group of nematodes that parasitize insects and other arthropods, causing their death. Unlike phytoparasitic nematodes, EPNs do not harm plants but, on the contrary, are used as biological agents for controlling insect pests. The greatest practical importance is attached to representatives of the families Steinernematidae and Heterorhabditidae (order Rhabditida), which are in symbiosis with bacteria of the genera Xenorhabdus and Photorhabdus, respectively. This symbiosis allows nematodes to quickly kill host insects (within 24–48 hours) and multiply efficiently in the cadaver (Grewal, 1999; Vashisth et al., 2013).

EPNs have a number of advantages over chemical insecticides: they are highly specific, safe for non-target organisms, do not pollute the environment, and can be used in integrated plant protection systems. However, their use is limited by sensitivity to UV radiation, low humidity, high temperatures, and production cost (Vashisth et al., 2013; Grewal, 1999).

In this chapter we will consider the systematics, biology, symbiosis with bacteria, life cycle, host-seeking strategies, production and application methods, and ecology of EPNs.

7.2. Systematics and Phylogeny of EPNs

Entomopathogenic nematodes belong to several taxonomic groups within the phylum Nematoda, but two families are of greatest practical importance:

• Steinernematidae (genus Steinernema) — order Rhabditida, suborder Tylenchina (according to the modern system — Rhabditina). Includes more than 80 described species (Grewal, 1999; Vashisth et al., 2013).
• Heterorhabditidae (genus Heterorhabditis) — order Rhabditida, suborder Rhabditina. Includes about 20 species (Grewal, 1999; Vashisth et al., 2013).

In addition, EPNs include representatives of other groups:

• Mermithidae (order Mermithida, class Enoplea) — parasites of insects, but their mass production is difficult, and they are rarely used in biocontrol (Grewal, 1999).
• Aphelenchoididae (order Aphelenchida) — some species (Bursaphelenchus spp.) are associated with insect vectors but are not true EPNs (Grewal, 1999).
• Tylenchida — some species (Deladenus siricidicola) parasitize insects (Grewal, 1999).

Modern molecular phylogenetic studies confirm the monophyly of Steinernematidae and Heterorhabditidae, but their relationships with other Rhabditida remain under study. It is believed that EPNs originated from free-living bacterivorous ancestors that acquired the ability to use insects as a habitat and food source (Schierwater, 2022; Grewal, 1999).

7.3. Symbiosis with Bacteria

A unique feature of Steinernematidae and Heterorhabditidae is their obligate mutualistic symbiosis with entomopathogenic bacteria:

• Steinernema spp. are symbiotic with bacteria of the genus Xenorhabdus (family Enterobacteriaceae) (Grewal, 1999; Vashisth et al., 2013).
• Heterorhabditis spp. are symbiotic with bacteria of the genus Photorhabdus (family Enterobacteriaceae) (Grewal, 1999; Vashisth et al., 2013).

Bacteria are localized in a specialized vesicle (bacterial pocket) in the intestine of the infective juvenile (IJ) — the infective third-stage juvenile, which is the only free-living stage in the EPN life cycle. The IJ does not feed but retains bacteria for several months in a resting state (Grewal, 1999; Vashisth et al., 2013).

When the IJ enters the insect body, bacteria are released into the hemocoel, where they rapidly multiply and cause septicemia. Bacteria produce a wide range of toxins and exoenzymes that kill the insect within 24–48 hours, as well as antibiotics that suppress growth of competing microorganisms. The insect cadaver turns into a nutrient medium for nematode reproduction (Grewal, 1999; Vashisth et al., 2013).

Interestingly, insects killed by Xenorhabdus spp. acquire a grayish or brownish color, whereas insects killed by Photorhabdus spp. become reddish, brown, or greenish and glow in the dark (luminescence) (Grewal, 1999; Vashisth et al., 2013).

7.4. Life Cycle and Infection Process

The life cycle of Steinernematidae and Heterorhabditidae includes the following stages:

1. Free-living infective juvenile (IJ) — third juvenile stage, which leaves the insect cadaver and actively searches for a new host. The IJ does not feed and uses lipid and glycogen reserves. The lifespan of the IJ depends on temperature and humidity but usually ranges from several weeks to several months (Grewal, 1999; Vashisth et al., 2013).

2. Penetration into the insect — the IJ enters the host body through natural openings (mouth, anus, spiracles) or through thin areas of the cuticle. Steinernema glaseri mainly penetrates through the intestine, whereas Heterorhabditis bacteriophora — through intersegmental membranes of the cuticle (Vashisth et al., 2013; Wang & Gaugler, 1999).

3. Release of bacteria — once in the hemocoel, the IJ releases symbiotic bacteria, which begin to multiply rapidly.

4. Death of the insect — bacteria kill the host within 24–48 hours, producing toxins and exoenzymes.

5. Reproduction of nematodes — nematodes feed on bacteria and decomposing insect tissues, passing through several generations. In Steinernematidae, the first generation is amphimictic (males and females); in Heterorhabditidae, the first generation is represented by hermaphroditic females, and subsequent generations are amphimictic (Grewal, 1999; Vashisth et al., 2013).

6. Emergence of new IJs — when food resources are depleted, juveniles develop into IJs, which leave the insect cadaver in search of a new host. Hundreds of thousands of IJs can emerge from one cadaver (Grewal, 1999; Vashisth et al., 2013).

An important difference between the families: for infection, a single IJ of Heterorhabditis is sufficient to cause infection, whereas for Steinernema at least two IJs (male and female) are required, since the first generation is dioecious (Grewal, 1999).

7.5. Host-Seeking Strategies

EPNs demonstrate two main host-seeking strategies (Grewal, 1999; Vashisth et al., 2013; Lewis, 2002):

• “Ambushers” — IJs remain motionless in soil, raising the anterior part of the body vertically (“J”-shaped posture), and wait for an insect to approach. They attack mobile surface insects. A typical representative is Steinernema carpocapsae (Grewal, 1999; Vashisth et al., 2013).

• “Cruisers” — IJs actively move in soil searching for a host, orienting to chemical signals (CO₂, volatiles). They are effective against sedentary or cryptic insects. Typical representatives — Steinernema glaseri, Heterorhabditis bacteriophora (Grewal, 1999; Vashisth et al., 2013).

• Intermediate strategies — some species (Steinernema feltiae, S. riobrave) combine elements of both strategies (Grewal, 1999).

The choice of strategy influences the effectiveness of control of particular pests. For example, for controlling soil pests inhabiting deep soil layers, “cruisers” are preferable, whereas for surface pests — “ambushers” (Vashisth et al., 2013; Lewis, 2002).

7.6. Hosts and Target Pests

EPNs have a wide host range under laboratory conditions, but under field conditions their spectrum is more limited. Larvae of Lepidoptera (cutworms, pyralids, codling moths) and Coleoptera (click beetles, scarabs, weevils) are most susceptible to EPNs (Grewal, 1999; Vashisth et al., 2013). Table 1 (Vashisth et al., 2013) provides an extensive list of target pests, including:

• Lepidoptera: Spodoptera litura (tobacco cutworm), Helicoverpa armigera (cotton bollworm), Chilo suppressalis (rice stem borer), Plutella xylostella (diamondback moth), Carposina niponensis (peach fruit moth).
• Coleoptera: Popillia japonica (Japanese beetle), Diabrotica virgifera (western corn rootworm), Rhynchophorus ferrugineus (red palm weevil), Otiorhynchus spp.
• Diptera: Bradysia spp. (fungus gnats), Liriomyza spp. (leafminer flies).
• Others: termites, cockroaches, ants.

EPNs are also used against cryptic pests (e.g., larvae living inside stems or tubers), where chemical insecticides are ineffective (Vashisth et al., 2013; Grewal, 1999).

7.7. Production and Formulation

7.8. Application and Factors Affecting Efficiency

7.9. Natural Distribution and Ecology

EPNs are obligate parasites of insects, but their IJs can exist in soil as free-living organisms for a long time. They are distributed everywhere, occurring in a variety of habitats: from cultivated fields and forests to deserts and beaches. In surveys, EPNs are found in 2–45% of soil samples (Vashisth et al., 2013; Hominick, 2002).

However, natural EPN abundance is usually low, and they rarely cause epizootics among insects. This is due to competition from other soil organisms (free-living nematodes, fungi, bacteria), limited hosts, and sensitivity to abiotic factors (Campos-Herrera et al., 2015; Vashisth et al., 2013).

Studies in Swiss soils (Campos-Herrera et al., 2015) showed that in agrocoenoses EPNs occur in trace amounts, and their activity is suppressed by competition with free-living nematodes (Acrobeloides group, Oscheius spp.) and predatory fungi. This indicates that in intensively cultivated soils the augmentation strategy (additional application) is more promising than conservation (preservation of natural populations) (Campos-Herrera et al., 2015).

7.10. Advantages and Limitations of EPNs

7.11. Commercial Preparations

More than 12 EPN species have been commercialized in various preparations on the international market (Vashisth et al., 2013, Table 2). The most common:

• Steinernema carpocapsae — Ortho Biosafe, Bio Vector, Exhibit, Nemassys, Stealth
• Steinernema feltiae — Entonem, Vector MG
• Steinernema riobrave — Bio Vector
• Steinernema scapterisci — Vector TL
• Heterorhabditis bacteriophora — Green Commandos, Soil Commandos
• Heterorhabditis megidis — Nemassys
• Heterorhabditis indica — Soil Commandos

In Russia and CIS countries, biopreparations based on EPNs are also registered, although their range is still limited (Vashisth et al., 2013; Akhatov et al., 2013).

8.1. Introduction

Nematodes occupy a unique position in agronomy: on the one hand, phytoparasitic forms cause enormous damage to agricultural production; on the other, free-living and entomopathogenic nematodes play an important role in maintaining soil health and biological control of pests. Understanding the practical importance of nematodes is necessary for developing effective and sustainable plant protection systems, as well as for assessing the state of soil ecosystems. In this chapter we will consider economic losses from phytoparasitic nematodes, quarantine measures, agrotechnical, chemical, and biological management methods, integrated plant protection, monitoring of abundance and species composition, and the practical importance of beneficial nematodes.

8.2. Economic Importance of Phytoparasitic Nematodes

Phytoparasitic nematodes are one of the most economically significant groups of agricultural crop pests. It is estimated that annual global crop losses from nematodes amount to about 11% for major food crops and more than 173 billion USD per year (Agrios, 2005; Caliskan et al., 2023). For individual crops and regions, losses can be catastrophic. For example, potato cyst nematodes Globodera rostochiensis and G. pallida cause losses of up to 9% of world potato production, and in some countries — up to 50–80% (Caliskan et al., 2023). Root-knot nematodes Meloidogyne spp. affect more than 3,000 plant species and can reduce yields of tomatoes, cucumbers, peppers, and potatoes by 30–70% (Reddy, 2019). The soybean cyst nematode Heterodera glycines annually causes damage estimated at 15.96 billion USD in ten countries producing 97% of the world soybean crop (Perry, 2013). The sugar beet cyst nematode Heterodera schachtii reduces sugar beet yield by 25–50% or more (Agrios, 2005; Perry, 2013). The stem nematode Ditylenchus dipsaci affects about 500 plant species, including onion, garlic, narcissus, clover, and can cause losses of up to 50–80% (Agrios, 2005; Perry, 2013). Root nematodes Pratylenchus spp. reduce yields of cereals, vegetables, and fruits by 30–50% (Caliskan et al., 2023; Perry, 2013). The burrowing nematode Radopholus similis causes “toppling disease” of bananas; losses can reach 40–70% (Perry, 2013). The citrus nematode Tylenchulus semipenetrans causes “slow decline” of citrus, reducing yield and tree lifespan (Agrios, 2005). The pine nematode Bursaphelenchus xylophilus destroys pine forests in Asia and Europe, causing enormous economic and ecological damage (Perry, 2013; Hockland et al., 2013).

In addition to direct crop losses, phytoparasitic nematodes increase production costs: the need for nematicides, quarantine measures, use of resistant varieties, crop rotations, and other methods. They also contribute to the development of complexes with other pathogens (fungi, bacteria, viruses), which enhances harmfulness (Agrios, 2005; Perry, 2013). For example, Pratylenchus penetrans in complex with Verticillium dahliae causes “early dying” of potato (Caliskan et al., 2023). Nematode vectors (Xiphinema, Longidorus, Trichodorus) transmit dangerous viruses such as grapevine fanleaf virus, tobacco ringspot virus, and pea early-browning virus (Perry, 2013; Decraemer & Geraert, 2013).

8.3. Quarantine Measures and Certification of Planting Material

Quarantine measures are a key element of protection against introduction and spread of dangerous phytoparasitic nematodes. International standards (ISPM, EPPO) regulate the movement of plants, soil, wood, and other materials that may be carriers of nematodes (Hockland et al., 2013; EPPO, 2013). Quarantine species such as Bursaphelenchus xylophilus, Globodera rostochiensis, G. pallida, Meloidogyne chitwoodi, M. fallax, M. enterolobii, Radopholus similis, Xiphinema americanum sensu lato are subject to mandatory diagnostics and control (Hockland et al., 2013; Caliskan et al., 2023).

Certification of planting material is an important measure to prevent the spread of nematodes. It includes:

• growing plants in sterile or nematode-free substrates;
• regular inspections and laboratory analyses;
• thermal treatment (hot water) of tubers, bulbs, cuttings;
• use of tissue cultures to obtain virus-free and nematode-free material (Agrios, 2005; Perry, 2013; Hockland et al., 2013).

For example, for bananas, tissue culture is used; for potatoes, certified seed tubers; for citrus, treatment of seedlings with hot water (50 °C, 10 min) (Perry, 2013). Certification reduces the risk of introducing nematodes into new regions and decreases crop losses.

8.4. Agrotechnical Methods

Agrotechnical methods are the basis of integrated plant protection against nematodes. They include:

• Crop rotation — alternating susceptible crops with non-host or resistant ones. Effective against nematodes with a narrow host range (e.g., Globodera spp., Heterodera glycines, Ditylenchus dipsaci). For Globodera, a 7–8-year rotation is recommended; for H. glycines — 1–2 years of non-host crops (Caliskan et al., 2023; Perry, 2013; Reddy, 2019). However, for polyphagous species (Meloidogyne, Pratylenchus), crop rotation is ineffective due to their wide host range (Caliskan et al., 2023).

• Resistant varieties — the most economical and environmentally friendly method. Resistance genes have been identified for many crops: Mi in tomato (against Meloidogyne), Rmc-1 in potato (against M. chitwoodi), H1 in potato (against G. rostochiensis), Rha1, Rha2, Rha3 in barley (against H. avenae), Rhg1–Rhg4 in soybean (against H. glycines) (Perry, 2013; Caliskan et al., 2023). However, prolonged use of resistant varieties can lead to selection of virulent populations, so rotation of resistance genes is necessary (Perry, 2013).

• Sowing dates — early sowing or planting allows plants to strengthen before mass nematode reproduction. For example, early potato planting reduces damage by G. rostochiensis (Caliskan et al., 2023; Reddy, 2019).

• Trap crops — plants that stimulate hatching of juveniles from cysts but do not support nematode reproduction. Example: Solanum sisymbriifolium for Globodera spp. (Caliskan et al., 2023; Reddy, 2019).

• Green manures and organic fertilizers — some plants (marigolds Tagetes spp., crotalaria Crotalaria spp., mustard, rapeseed) release nematicidal compounds (thiophenes, glucosinolates) or stimulate antagonistic microflora (Caliskan et al., 2023; Reddy, 2019; Weil, 2017). Application of organic matter increases soil suppressiveness (Weil, 2017).

• Soil solarization — heating soil under transparent film to 50–60 °C; effective in hot climates (Caliskan et al., 2023; Reddy, 2019).

• Flooding — prolonged flooding (17 weeks) reduces abundance of Ditylenchus dipsaci (Caliskan et al., 2023; Reddy, 2019).

• Destruction of plant residues — removal and burning of affected plants reduces the infection background (Reddy, 2019; Akhatov et al., 2013).

8.5. Chemical Methods

Chemical nematicides remain an important tool in intensive agriculture, especially for high-value crops (vegetables, flowers, potatoes, tobacco). They are divided into:

• Fumigants — 1,3-dichloropropene (1,3-D), metam sodium, dazomet, methyl bromide (banned in the EU), chloropicrin. Applied by injection into soil under film (Perry, 2013; Agrios, 2005).
• Non-fumigants — carbamates (aldicarb, carbofuran, oxamyl), organophosphates (fenamiphos, ethoprophos, fensulfothion). Act systemically or contactly, less phytotoxic (Perry, 2013; Reddy, 2019).

However, the use of nematicides is limited due to:

• high toxicity to humans and the environment;
• accumulation in soil and groundwater;
• development of resistance in nematodes;
• bans on many preparations (methyl bromide, aldicarb, carbofuran) in the EU and other countries (Caliskan et al., 2023; Perry, 2013).

Modern trends — transition to less toxic nematicides (fluopyram, fluensulfone), biopreparations, and integrated methods (Caliskan et al., 2023; Thoden et al., 2019).

8.6. Biological Methods

Biological control of nematodes is a promising direction based on the use of natural enemies: predatory fungi, bacteria, predatory nematodes, protozoa, mites, collembolans. The greatest practical application has been obtained by:

• Predatory fungi — Arthrobotrys spp., Dactylella spp. form traps (adhesive networks, rings, knobs) for nematodes (Weil, 2017; Reddy, 2019).
• Parasitic fungi — Pochonia chlamydosporia, Purpureocillium lilacinum infect eggs and females of cyst and root-knot nematodes. Commercial preparations have been created on their basis (BioAct, PL Plus) (Perry, 2013; Caliskan et al., 2023).
• Bacteria — Pasteuria penetrans parasitizes juveniles of Meloidogyne, Pratylenchus, Heterodera. It forms endospores that persist in soil for years. Commercial preparation Econem (Caliskan et al., 2023; Perry, 2013).
• Entomopathogenic nematodes — Steinernema and Heterorhabditis are used to control insect pests, but not phytoparasitic nematodes (Vashisth et al., 2013; Grewal, 1999).
• Mycorrhizal fungi — arbuscular mycorrhizae (Glomus spp.) increase plant resistance to nematodes by competing for infection sites and improving plant nutrition (Weil, 2017; Reddy, 2019).
• Organic amendments — application of composts, manure, green manures increases soil suppressiveness and reduces nematode abundance (Weil, 2017; Caliskan et al., 2023).

Biological methods are environmentally friendly, but their effectiveness varies depending on soil and climatic conditions, nematode species, and strain of the agent. They work better in integrated systems (Perry, 2013; Caliskan et al., 2023).

8.7. Integrated Pest Management (IPM)

Integrated Pest Management (IPM) is a strategy combining agrotechnical, biological, chemical, and quarantine methods to maintain nematode abundance below the economic threshold of harmfulness. IPM includes:

• monitoring nematode abundance and species composition;
• determining economic thresholds of harmfulness;
• selection of resistant varieties and crop rotations;
• use of biopreparations and organic fertilizers;
• application of nematicides only when necessary and in minimal doses;
• quarantine and certification measures (Perry, 2013; Caliskan et al., 2023; Reddy, 2019).

Examples of successful IPM: control of Globodera on potato (crop rotation + resistant varieties + nematicides), control of Meloidogyne on vegetables (grafting onto resistant rootstocks + biopreparations + solarization), control of Radopholus similis on bananas (tissue cultures + hot water + nematicides) (Caliskan et al., 2023; Perry, 2013).

8.8. Monitoring of Abundance and Species Composition

Monitoring is the basis for decision-making in IPM. It includes:

• sampling of soil and plant samples;
• extraction of nematodes (Baermann method, flotation, centrifugal flotation, elutriation);
• morphological and molecular identification;
• quantitative counting (number of individuals per 100 cm³ of soil or per 1 g of roots);
• calculation of indices (maturity index, PPI, EI, SI) to assess soil condition (EPPO, 2013; Huang, 2012; Weil, 2017).

Modern molecular methods (qPCR, LAMP, metabarcoding) allow rapid and accurate determination of species and their abundance, which is especially important for quarantine species (Caliskan et al., 2023; Campos-Herrera et al., 2015). Economic thresholds of harmfulness have been established for many crops: for example, for Meloidogyne on tomatoes — 1–2 juveniles per 100 cm³ of soil; for Globodera on potato — 1–2 eggs/g of soil (Caliskan et al., 2023; Perry, 2013).

8.9. Practical Importance of Free-Living and Entomopathogenic Nematodes

Free-living soil nematodes are indicators of soil health. Their communities are used to assess the impact of agricultural practices (tillage, fertilizers, pesticides), restoration of disturbed lands, and pollution monitoring (Huang, 2012; Weil, 2017). High diversity and presence of cp-4, cp-5 species indicate a stable ecosystem; dominance of cp-1, cp-2 — disturbance (Bongers, 1990; Weil, 2017).

Entomopathogenic nematodes are biological insecticides. They are effective against soil and cryptic pests (cutworms, wireworms, weevils, leafminer flies), safe for non-target organisms, and compatible with agrochemicals. Commercial preparations based on Steinernema and Heterorhabditis are registered in many countries (Vashisth et al., 2013; Grewal, 1999). In Russia, biopreparations based on EPNs are also being developed (Akhatov et al., 2013).

9.1. Nematodes — A Diverse and Ecologically Important Group

Nematodes represent one of the most numerous, diverse, and ecologically significant groups of multicellular animals on Earth. They belong to the separate phylum Nematoda within the clade Ecdysozoa and are characterized by a unique combination of a conservative basic body plan with enormous species diversity and ecological plasticity (Schierwater, 2022; Perry, 2013). Nematodes inhabit almost all possible habitats — from Arctic ice to tropical forests, from deep-ocean trenches to high mountains, from freshwater bodies to saline soils. Their abundance in soil reaches 10^5–107 individuals per m^{2, and biomass — 1–30 g/m}2 (Weil, 2017). The total number of described species exceeds 28,000, although actual diversity may be orders of magnitude higher (Schierwater, 2022).

Nematodes play a key role in soil food webs, participating in decomposition of organic matter and cycling of biogenic elements. They occupy several trophic levels — from bacterivores and fungivores to predators and omnivores — and serve as indicators of soil condition and anthropogenic load (Huang, 2012; Weil, 2017). Free-living soil nematodes such as Caenorhabditis elegans have become important model objects in biology, genetics, neuroscience, and toxicology (Schierwater, 2022; Reddy, 2019).

9.2. Dual Role of Nematodes in Agronomy

In agronomy, nematodes play a dual role. On the one hand, phytoparasitic forms cause significant damage to agricultural production. Annual global crop losses from nematodes are estimated at 11% for major food crops and more than 173 billion USD (Agrios, 2005; Caliskan et al., 2023). Cyst nematodes (Heterodera, Globodera), root-knot nematodes (Meloidogyne), stem nematodes (Ditylenchus dipsaci), leaf nematodes (Aphelenchoides), root nematodes (Pratylenchus, Radopholus), ectoparasitic nematodes (Xiphinema, Longidorus, Trichodorus), and other groups affect almost all agricultural crops, causing stunted growth, chlorosis, wilting, reduced yield and product quality (Perry, 2013; Agrios, 2005). Nematode vectors transmit dangerous viruses such as grapevine fanleaf virus and tobacco ringspot virus (Decraemer & Geraert, 2013; Perry, 2013). Phytoparasitic nematodes also form complexes with fungi, bacteria, and other pathogens, enhancing harmfulness (Caliskan et al., 2023; Agrios, 2005).

On the other hand, free-living and entomopathogenic nematodes are beneficial. Free-living soil nematodes participate in decomposition of organic matter, mineralization of nitrogen and phosphorus, regulate the abundance of bacteria and fungi, and serve as indicators of soil health (Huang, 2012; Weil, 2017). Entomopathogenic nematodes Steinernema and Heterorhabditis are used for biological control of insect pests. They are symbiotic with bacteria Xenorhabdus and Photorhabdus, which kill insects within 24–48 hours, and are used against a wide range of soil and cryptic pests (Vashisth et al., 2013; Grewal, 1999). EPNs are safe for non-target organisms, compatible with agrochemicals, and can be used in integrated plant protection systems (Vashisth et al., 2013; Grewal, 1999).

9.3. Systematics and Identification — Basis for Management

Accurate identification of nematodes is a necessary basis for phytosanitary diagnostics, quarantine measures, harmfulness forecasting, and selection of effective protection measures. Modern nematode systematics is based on an integrative approach combining morphological, morphometric, biochemical, and molecular-genetic methods (Perry, 2013; EPPO, 2013). Molecular methods (PCR, qPCR, LAMP, sequencing, metabarcoding) allow reliable distinction of closely related species, detection of cryptic species and species complexes, and detection of nematodes in mixed populations and in soil without preliminary extraction (Caliskan et al., 2023; Campos-Herrera et al., 2015). For quarantine species, standardized EPPO protocols have been developed, including morphological and molecular methods (EPPO, 2013; Hockland et al., 2013).

The problem of cryptic species and species complexes remains relevant. Examples: Meloidogyne spp., Pratylenchus spp., Globodera spp., Radopholus similis / R. citrophilus, Bursaphelenchus xylophilus / B. mucronatus (Perry, 2013; Caliskan et al., 2023). Their differentiation requires comprehensive studies including morphometrics, isoenzyme analysis, molecular markers, and crossing experiments.

9.4. Integrated Pest Management — Strategy for the Future

Integrated Pest Management (IPM) is a strategy combining agrotechnical, biological, chemical, and quarantine methods to maintain nematode abundance below the economic threshold of harmfulness. IPM includes monitoring nematode abundance and species composition, determining economic thresholds, selecting resistant varieties and crop rotations, using biopreparations and organic fertilizers, applying nematicides only when necessary and in minimal doses, as well as quarantine and certification measures (Perry, 2013; Caliskan et al., 2023; Reddy, 2019).

Examples of successful IPM: control of Globodera on potato (crop rotation + resistant varieties + nematicides), control of Meloidogyne on vegetables (grafting onto resistant rootstocks + biopreparations + solarization), control of Radopholus similis on bananas (tissue cultures + hot water + nematicides) (Caliskan et al., 2023; Perry, 2013). Biological methods such as predatory fungi (Arthrobotrys), parasitic fungi (Pochonia chlamydosporia, Purpureocillium lilacinum), bacteria (Pasteuria penetrans), mycorrhizal fungi, and organic amendments are becoming increasingly important in sustainable agroecosystems (Perry, 2013; Weil, 2017; Reddy, 2019). Chemical nematicides remain a tool for high-value crops, but their use is declining due to toxicity and bans (Caliskan et al., 2023; Thoden et al., 2019).

9.5. Practical Importance and Monitoring

Monitoring of nematode abundance and species composition is the basis for decision-making in IPM. It includes sampling of soil and plant samples, extraction of nematodes (Baermann method, flotation, centrifugal flotation, elutriation), morphological and molecular identification, quantitative counting, and calculation of indices (maturity index, PPI, EI, SI) to assess soil condition (EPPO, 2013; Huang, 2012; Weil, 2017). Economic thresholds of harmfulness have been established for many crops: for example, for Meloidogyne on tomatoes — 1–2 juveniles per 100 cm³ of soil; for Globodera on potato — 1–2 eggs/g of soil (Caliskan et al., 2023; Perry, 2013). Modern molecular methods (qPCR, LAMP, metabarcoding) allow rapid and accurate determination of species and their abundance, which is especially important for quarantine species (Caliskan et al., 2023; Campos-Herrera et al., 2015).

Free-living soil nematodes are used as bioindicators of soil condition and anthropogenic load. Their communities respond to tillage, fertilization, pesticides, heavy metal contamination, changes in pH, moisture, and temperature (Huang, 2012; Weil, 2017). Maturity index and other ecological indices allow assessment of the sustainability and health of soil ecosystems (Bongers, 1990; Weil, 2017).

9.6. Prospects and Challenges

Despite significant progress in the study of nematodes, many questions remain open. The phylogeny of Enoplia, the position of some families (e.g., Trichodoridae), boundaries of genera and species, and mechanisms of the evolution of parasitism all require further research (Schierwater, 2022; Perry, 2013). The problem of cryptic species and species complexes remains relevant and requires an integrative approach (Caliskan et al., 2023; Perry, 2013). Climate change may lead to expansion of phytoparasitic nematode ranges into colder regions, creating new threats to agriculture (Caliskan et al., 2023; Perry, 2013). Development of resistance to nematicides and selection of virulent populations on resistant varieties require constant monitoring and rotation of control methods (Perry, 2013; Caliskan et al., 2023).

Promising directions:

• development of new biopreparations based on antagonistic fungi, bacteria, and predatory nematodes;
• use of RNA interference (RNAi) to suppress nematode virulence genes;
• genetic modification of plants to increase resistance;
• precision agriculture and remote sensing for nematode monitoring;
• integration of molecular methods into routine diagnostics;
• study of the soil microbiome and its role in suppressiveness to nematodes (Caliskan et al., 2023; Perry, 2013; Schierwater, 2022).

9.7. Concluding Remarks

Nematodes are an amazing group of organisms, combining simplicity of the basic body plan with enormous diversity and ecological plasticity. In agronomy they play a dual role: phytoparasitic forms cause significant damage to agriculture, while free-living and entomopathogenic nematodes are beneficial, participating in maintaining soil health and biological control of pests. Accurate identification, monitoring, and understanding of nematode biology are the key to effective and sustainable plant protection. Integrated pest management, combining agrotechnical, biological, chemical, and quarantine methods, reduces dependence on nematicides and increases the sustainability of agroecosystems. Further research should be aimed at developing new environmentally safe control methods, studying molecular mechanisms of parasitism and symbiosis, and training qualified nematology specialists capable of solving practical plant protection problems under changing climate and growing requirements for sustainable agriculture.

Nematodes are not only “invisible enemies” of plants but also the most important component of soil biota, indicators of soil health, and potential allies of humans in the struggle for sustainable agriculture. Their study continues to be one of the most relevant and promising areas of modern biology and agronomy.