Nematode-Trapping Fungi

ABSTRACT

Nematode-trapping fungi are a unique and intriguing group of carnivorous microorganisms that can trap and digest nematodes by means of specialized trapping structures. They can develop diverse trapping devices, such as adhesive hyphae, adhesive knobs, adhesive networks, constricting rings, and nonconstricting rings. Nematode-trapping fungi have been found in all regions of the world, from the tropics to Antarctica, from terrestrial to aquatic ecosystems. They play an important ecological role in regulating nematode dynamics in soil. Molecular phylogenetic studies have shown that the majority of nematode-trapping fungi belong to a monophyletic group in the order Orbiliales (Ascomycota). Nematode-trapping fungi serve as an excellent model system for understanding fungal evolution and interaction between fungi and nematodes. With the development of molecular techniques and genome sequencing, their evolutionary origins and divergence, and the mechanisms underlying fungus-nematode interactions have been well studied. In recent decades, an increasing concern about the environmental hazards of using chemical nematicides has led to the application of these biological control agents as a rapidly developing component of crop protection.

INTRODUCTION

There are about 700 species of taxonomically diverse fungi that are be able to attack living nematodes (juveniles, adults, and eggs) and use them as a nutrient source (1, 2). The most important genera include Purpureocillium, Pochonia, Hirsutella, Nematophthora, Arthrobotrys, Drechmeria, Fusarium, and Dactylellina (3, 4). Among these nematophagous fungi, only a few species are obligate parasites of nematodes, but the majority are facultative saprophytes (5–7). Based on the mechanisms by which they attack nematodes, these nematophagous fungi are usually divided into four general groups: (i) nematode-trapping fungi that use specialized trapping structures differentiated from hyphae; (ii) endoparasitic fungi that use their spores; (iii) the opportunistic fungi that invade or colonize nematode eggs, females, or cysts with their hyphal tips; and (iv) toxin-producing fungi that immobilize nematodes before invasion (1, 8). Nematophagous fungi are important natural enemies of nematodes in soil ecosystems. In recent decades, environmental and health concerns over the use of chemical nematicides have greatly increased the demand for the development of biological control agents in plant protection. The reason for the growing interest in nematophagous fungi is mostly their potential as biocontrol agents against plant- and animal-parasitic nematodes. So far, a substantial number of myconematocides have been developed worldwide (3, 4, 6).

Nematode-trapping fungi are soilborne fungi that entrap free-living nematodes by using trapping structures. These fungi have evolved sophisticated trapping structures, including constricting rings and five types of adhesive traps (sessile adhesive knobs, stalked adhesive knobs, adhesive nets, adhesive columns, and nonconstricting rings) (9–12). About 380 species of nematode-trapping fungi have been reported from different regions of the world (2). Different fungal species can produce one or more types of different trapping devices (11, 12). Most nematode-trapping fungi can live both saprophytically on organic matter and as predators by capturing tiny animals (7, 13–16). Traps are not only the weapons that nematode-trapping fungi use to attack nematodes, but are also an important indicator of their switch from a saprophytic to a predacious lifestyle (10, 16). Nematode-trapping fungi are usually not host specific and can trap all soil-dwelling nematodes (1, 17, 18). With increasing discovery of the corresponding sexual-asexual morph connections, most nematode-trapping fungi are known as asexual morphs of Orbilia spp. within the Orbiliales (Ascomycota) (2, 19, 20). Recent analyses based on DNA sequences suggest that trapping devices provide the most relevant morphological features for taxonomic classification of predatory asexual Orbiliaceae (11, 21–23). These fungi have been classified into four genera: Arthrobotrys (adhesive three-dimensional networks), Dactylellina (stalked adhesive knobs and/or nonconstricting rings), Drechslerella (constricting rings), and Gamsylella (adhesive branches and unstalked knobs). However, given the new nomenclatural regulations that employ only one name per fungus, the names for these nematode-trapping fungi have to be revised (24, 25).

Previous studies of nematode-trapping fungi mainly focused on investigation of their species diversity and abundance in different regions and habitats, taxonomy and phylogeny, laboratory screening, and field trials for nematode control (4, 6, 15, 26–31). In recent years, more attention has been paid to the molecular mechanisms underlying the process of fungi trapping and infecting nematodes, the origin and evolution of trapping structures, genomics, and proteomics (10, 11, 32–37). Virulent factors, the role of proteases, chitinases and small chemical molecules, and the regulation of trap formation have been extensively studied with modern techniques (16, 37–40). There is an increasing body of evidence to illustrate that the predatory lifestyle of nematode-trapping fungi has evolved among cellulolytic or lignolytic fungi as a response to nutrient deficiencies in nitrogen-limiting habitats. Based on molecular clock and fossil evidence, nematode-predatory Orbiliaceae were estimated to have originated as a result of mass extinctions in the Permian and Triassic (10). Recent studies also showed that nematophagous fungi can detect and respond to ascarosides, which are an evolutionarily highly conserved family of small molecules secreted by many species of soil-dwelling nematodes as molecular signal to recognize prey and trigger trap formation (37). By using comparative genomics and transcriptomics, scientists deciphered two genomic mechanisms leading to the evolution of parasitism in nematode-trapping fungi: the formation of novel genes through gene duplication and the differential regulation of existing genes identified by differential expression of orthologous genes (34, 35). Understanding the molecular and genomic basis of nematode-trapping fungi and nematode interactions provides crucial insights for developing effective biological control methods against plant-parasitic nematodes.

TAXONOMY AND DIVERSITY

Nematode-trapping fungi are a taxonomically heterogeneous group of organisms (distributed in Ascomycota, Basidiomycota, and Zygomycota) that can use these special structures (traps) to capture free-living nematodes in soil (1, 17, 41, 42). About 380 species of nematode-trapping fungi have been reported from different regions of the world (2), including species in the genera Arthrobotrys, Cystopage, Dactylellina, Dactylella, Drechslerella, Hohenbuehelia, Hyphoderma, Monacrosporium, Nematoctonus, Orbilia, Stylopage, Tridentaria, Triposporina, and Zoophagus. Orbiliaceous members represent the largest group of nematode-trapping fungi, which include about 96 species and are currently assigned to the asexual genera Arthrobotrys (53 species), Dactylellina (28 species), and Drechslerella (14 species) (17).

Molecular phylogenetic analyses indicate that the monophyletic classes Orbiliomycetes and Pezizomycetes are among the earliest diverging branches of Pezizomycotina, the largest subphylum of the Ascomycota (43–45). Orbiliomycetes is represented by a single order, Orbiliales, with the only family, Orbiliaceae, consisting of three accepted sexual genera, Hyalorbilia, Orbilia, and Pseudorbilia (46, 47). This family has about 288 described species (19). The members of Orbiliaceae are characterized by small, translucent, waxy, disk-shaped apothecia that are formed in soil and wood. Apothecia are produced without stromata and have an ectal excipulum of globose, angular, or prismatic cells. Small asci with truncate to hemispherical apices are intermixed with paraphyses that typically are swollen at their tips. Small hyaline ascospores usually possess an apical spore body that selectively stains in cresyl blue (48). About 10 asexual genera have been connected with Orbiliaceae (49), and all are hyphomycetous. Certain of these asexual genera are commonly known nematode-trapping fungi, and their evolution and phylogeny have been studied with nuclear and protein-coding genes.

The first nematode-trapping fungus, Arthrobotrys superba as the type species of Arthrobotrys, was described in 1839, but its nematode-trapping habit was unknown until the observation of Arthrobotrys oligospora by Zopf in 1888; the connection between A. oligospora and Orbilia fimicola, and its nematode-trapping lifestyle, was confirmed by Pfister in 1994, 155 years after the first nematode-trapping fungus was described (50). The characteristics of the genus Arthrobotrys were considered to be hyaline conidiophores producing conidia asynchronously on short denticles at swollen conidiogenous heads or clusters of pronounced denticles, the conidia being subhyaline, obovoid, or clavate and (0–)1(–6)-septate (50). Trapping devices were considered to be constricting rings, adhesive nets, hyphae, or adhesive knobs (50). This wide generic concept based on reproductive structures resulted in considerable confusion, although since 1930, nematode-trapping fungi have been mostly assigned to Arthrobotrys, Dactylaria, Dactylella, and some other genera.

The trapping devices of nematode-trapping fungi are developed from the vegetative mycelia. The known trap types include adhesive networks, adhesive knobs, constricting rings, nonconstricting rings, adhesive branches, undifferentiated or unmodified adhesive hyphae, stephanocysts, spiny balls, and acanthocytes (51–54) (Fig. 1). Members of the Orbiliaceae can produce the first five types of traps (9). Zoophagus species of Zygomycetes (55, 56) and Nematoctonus species of Basidiomycetes can also produce adhesive knobs (57). Traps of adhesive hyphae are restricted to the genera Stylopage and Cystopage of Zygomycetes (58), while stephanocysts are restricted to the genus Hyphoderma of Basidiomycota (51). Spiny balls and acanthocytes are only formed, respectively, by Coprinus comatus (53, 54) and Stropharia rugosoannulata (59) in Agaricales of Basidiomycota. The sexual morphs of most nematode-trapping species are now assigned to Orbilia, and their type of trapping apparatus can be used as robust indicators of generic delimitation. These fungi were also classified according to their trap types with the support of genetic data as follows: Arthrobotrys (adhesive three-dimensional networks), Dactylellina (stalked adhesive knobs and/or nonconstricting rings), Drechslerella (constricting rings), and Gamsylella (adhesive branches and unstalked knobs) (17).

FIGURE 1.

Structures of traps in nematode-trapping fungi. (A, B) Adhesive networks of Arthrobotrys oligospora. Bar, 20 μm. (C–E) Constricting rings of Drechslerella stenobrocha. Bar, 20 μm. (F) Adhesive knobs and nonconstricting rings of Dactylellina haptotyla. Bar, 20 μm. (G) Nematode trapped by D. haptotyla. Bar, 40 μm. (H, I) Adhesive columns of Gamsylella cionopaga. Bar, 20 μm.

Fungal taxonomy has changed a lot in the past decades, from being based on only morphological observations to being a combined analysis of morphology and DNA sequence data. Morphology-based classification based on conidia was demonstrated to be inadequate in reflecting natural relationships among the nematode-trapping fungi. The first phylogenetic analysis of nematode-trapping fungi was conducted by Rubner in 1996. She used molecular data to rationalize the classification of nematode-trapping fungi based on types of trapping devices (60). Phylogenetic studies based on rDNA sequence analysis also found that trapping devices are more informative than morphological characters in delimiting genera (11, 21–23, 61). Subsequently, in 1999 Scholler et al. classified nematode-trapping fungi into four genera using 18S and internal transcribed spacer (ITS) rDNA analysis. Li et al. (21) re-evaluated the placement of nematode-trapping genera based on 28S, 5.8S, and β-tubulin analysis, and the establishment of Gamsylella proposed by Scholler et al. was criticized and rejected.

Although trapping devices have been shown to be vital in generic delimitation of nematode-trapping fungi, species in the same genus are mainly delimited from each other by the morphology of their conidia and conidiophores. With more and more connections between nematode-trapping fungi and Orbiliaceae being established (49, 50, 62–64), phylogenetic analysis including existing asexual species indicated that conidial morphology showed little correlation, while characters of their sexual morphs showed a high level of correlation. For example, Arthrobotrys belongs to series Auricolores, which is defined by the ability to form adhesive networks, while the asexual Trinacrium type belongs to species of Hyalorbilia and Orbilia subgenus Hemiorbilia and subgenus Orbilia (H.-O. Baral, personal communication). Further studies are needed to set up a natural classification system based on the connection of asexual and sexual nematode-trapping fungi. In 2012, the International Code of Nomenclature for algae, fungi, and plants was published, which no longer allows a single fungal species to have two names; the older name takes priority except in exceptional circumstances (24, 25). The asexual genus above that is linked to Orbilia sexual morphs may need to be allocated to Orbilia or, alternatively, to an asexual genus. Discussions are presently under way in the Orbiliomycetes working group of the International Commission on the Taxonomy of Fungi to adopt a single nomenclature for this group of fungi.

ECOLOGY AND EVOLUTION

Nematophagous fungi represent an important group of soil microorganisms that can suppress populations of plant and animal parasitic nematodes (3, 4, 26–28, 65–67). These fungi are broadly distributed in terrestrial and aquatic ecosystems, from the tropics to Antarctica (7, 13, 15, 29–31, 68–70). Efforts have frequently been made to control diseases caused by plant-parasitic nematodes by using nematode-trapping fungi, but results have been inconsistent. It has subsequently been pointed out that a fundamental knowledge of the ecology of these fungi is essential before the value of such biological control methods can be assessed. Environmental factors greatly affect the occurrence and activity of soil microorganisms. However, our knowledge about how the environment affects the abundance and activity of nematophagous fungi is still relatively poor (14, 31).

The effect of major biotic and abiotic variables such as soil moisture, organic matter, pH, nematode density, soil nutrients, and submerged water conditions on the distribution of nematode-trapping fungi has been extensively studied. Previous studies by Gray on the distribution of nematophagous fungi indicate that the occurrence of certain species and groups of fungi is associated with specific soil variables: in particular, pH, soil moisture, nutrients (N, P, K), heavy metal, and nematode density (15, 71). Gray revealed that soil nutrients such as N, P, and K were all positively correlated with nematode density. Based on his results, species with stalked, knobbed trapping devices (Dactylellina) and species with constricting rings (Drechslerella) were more frequently isolated from organically richer soils which contained a greater density of nematodes (31, 72, 73). However, net-forming species (Arthrobotrys) are largely independent of soil fertility, especially low K (74). In a recent survey, Mo et al. found that the diversity of nematode-trapping fungi was positively correlated with lead concentration (75). These soil variables are known to vary with depth, as are the densities of soil bacteria, fungi, and nematodes (76, 77), and a high level of nematode-capturing activity has been recorded from the rhizosphere area. However, there are also large variations depending on plant species and soil types (78). The species of nematode-trapping fungi vary with soil depth (79). Peterson and Katznelson (80) revealed that the greatest diversity occurred in the upper 10 to 30 cm of soils, and this was a positive correlation between the population density of nematophagous fungi and root-knot nematodes in peanut fields. Hao et al. (13) observed that the nematode-trapping fungi were not detected deeper than 4 m in a freshwater pond. Several studies have also been carried out on horizontal distribution (81). For example, Persson et al. (81) studied the growth and dispersion of A. superba under natural conditions determined by a radioactive tracing technique.

The number of nematode-trapping species present in a specific soil and their population densities can vary considerably. Application of chopped organic amendment and glucose to soil could increase the activity of nematode-trapping species perhaps because of the consequent increase in the number of free-living and microbivore nematodes (82–84). Although organic amendments probably stimulated population densities, similar population densities of trapping fungi were found in plots with and without organic amendments. The effect of abscisic acid and nitric oxide on the nematode-trapping fungus Drechslerella stenobrocha AS6.1 was tested, and it was demonstrated that the trap development and nematode-trapping capability of D. stenobrocha were increased by abscisic acid but decreased by nitric oxide (85). It is apparent that the trapping fungi need a carbohydrate source for their proliferation, but other factors, such as those that cause fungistasis, are also important for their abundance and trophic state in soil. It is hypothesized that Orbilia species, the sexual morph of Arthrobotrys species, are weak wood decomposers and that nematode cadavers act as an important supply of nitrogen. Predaceous behavior of A. oligospora can be controlled either by physiologically active compounds (amino acids or vitamins) present in nematodes or by nitrogen sources.

The majority of nematode-trapping fungi colonize soil, waiting until passing nematodes touch them. Some fungi increase their trapping chances by producing secondary attractive compounds for nematodes, such as A. superba, which attracts second-stage juveniles (J2) of Meloidogyne species (86). Others grow in the rhizosphere and can even colonize plant roots (76, 80, 87), which gives them superior predatory activity to trap plant-parasitic nematodes on their way to roots. For example, A. oligospora was more abundant in the rhizosphere of tomato and barley plants because of its chemotropical attraction to the root tips. Plant species obviously influence the rhizosphere and external root colonization. The highest incidence and diversity of nematode-trapping fungi were detected in association with rhizosphere of peas, while Dactylellina ellipsospora and Arthrobotrys dactyloides were successfully colonized on tomato roots in a pot experiment (87, 88).

Carbon and nitrogen are two essential nutrients for fungal growth and reproduction. It has been proposed that the nematode-trapping lifestyle is an evolutionary response by cellulolytic or lignin-degrading fungi to nutrient deficiencies in nitrogen-limiting habitats. Because nitrogen is essential for fungal growth and is not freely available in dead wood or in soil where carbon is abundant, direct capture of nitrogen compounds from free-living nematodes or other small animals is advantageous (89). Many network-forming species do not produce a network trap spontaneously, and they are more saprophytic than other nematode-trapping fungi. Formation of network-trapping devices can be induced by the presence of nematodes or substances of animal origin known as nemin. The adhesive network is a primitive characteristic induced only by covering the hypha with a thin film of stick fibrils. Fungal species with other types of trapping devices, such as adhesive knobs (sessile or stalked) and constricting rings, produce trapping devices spontaneously. The spontaneous trap formers more effectively prey on nematodes than do nonspontaneous formers, such as species forming adhesive networks, which have the flexibility to become more predacious by induction of more traps (10).

Various hypotheses on the origin and evolution of nematode-trapping lifestyles in the Ascomycota have been proposed, but conflicts exist between molecular and phenotypic phylogenies (10, 11, 21, 61). The phylogenetic relationships between the different nematode-trapping species need to be known to understand the evolution of parasitism in nematode-trapping fungi. Many molecular phylogenetic studies have shown that the largest group of nematode-trapping fungi belongs to a monophyletic group in the order Orbiliales (Ascomycota). A recent study based on a comprehensive phylogenetic analysis of the nucleotide sequences of three protein-coding genes and rDNA in the internal transcribed spacer region demonstrated that the trapping mechanisms within the Orbiliales have evolved along two major lineages, one leading to species with constricting rings and the other to species with adhesive traps, including three-dimensional networks, knobs, and branches. Among those adhesive trapping devices, the adhesive network separated itself from other trapping mechanisms at an early stage. The adhesive knob evolved through stalk elongation and finally developed into nonconstricting rings. It was shown that the derived adhesive traps are at a highly differentiated stage, but in this study the evolution of the adhesive traps was still ambiguous, partly because the number of taxa representing each type of trapping device was uneven (11).

In a more recent study, data based on a phylogeny of five protein-encoding genes and molecular clock calibration based on two fossil records revealed that fungal carnivorism diverged from saprophytism about 419 million years ago (Mya), which was after the origin of nematodes about 550 to 600 Mya. Active carnivores (fungi with constricting rings) and passive carnivores (fungi with adhesive traps) diverged from each other around 246 Mya, shortly after the occurrence of the Permian-Triassic extinction event about 251.4 Mya. The major adhesive traps evolved around 198 to 208 Mya, which was within the time frame of the Triassic-Jurassic extinction event about 201.4 Mya (Fig. 2). However, more evidence, including additional fossil records, is still needed to illustrate if fungal carnivore evolution was a response to mass extinction events. This study also illustrated a clearer route for the evolution of adhesive traps. Sessile adhesive knobs and an extinct trapping structure (unicellular ring) originated from the adhesive ancestor. Adhesive columns diverged from sessile adhesive knobs by the proliferation of adhesive knobs into a column, which increased the adhesive surface and predatory efficiency. The unicellular ring probably became extinct due to low predatory efficiency (the unicellular ring is morphologically similar to nonconstricting rings, which have the lowest predatory efficiency of existing trapping fungi). The unicellular ring evolved along two lineages to increase predatory efficiency. One formed adhesive nets by proliferating the ring into a network of rings. The other formed nonconstricting rings accompanied by stalked adhesive knobs, in which the single-celled rings developed into three-celled nonconstricting rings, unicellular knobs, and stalked adhesive knobs (10).

FIGURE 2.

Phylogeny of carnivorous Orbiliomycetes constructed by RAxML using five protein-coding genes. The bootstrap supports are marked on the branch. Trapping devices are drawn on the left.

MECHANISMS OF FUNGI TRAPPING NEMATODES

Plant-parasitic nematodes cause significant damage to a broad range of vegetables and agricultural crops throughout the world. Understanding the molecular basis of microbe-nematode interactions could lead to the development of more efficient and reliable methods for biological control of parasitic nematodes (12, 90, 91).

Nematode-trapping fungi infect their hosts through a sequence of events (1). They use specialized trapping devices to catch and consume nematodes, and previous studies demonstrated that most fungal species do not produce traps constitutively but, rather, initiate trap formation in response to their prey (92–94). Recognition and adhesion were the first steps in nematode-trapping fungi infecting their hosts. However, little is known about the molecular mechanisms of recognition and adhesion. Lectin has been reported to be involved in the recognition process (95–97). The finding of lectins in the role of nematode-fungi interaction came from an observation that the interaction between A. oligospora and nematodes was mediated by a GalNAc-(N-acetyl-d-galactosamine)-specific fungal lectin binding to receptors present on the nematode surface (97). Similar experiments have also indicated that lectins play a role in the adhesion to host surfaces by a number of parasitic and symbiotic fungi (98). However, after a gene encoding such a lectin was deleted in A. oligospora by homologous recombination, the deletion mutant showed little decrease in spore (conidia) germination, saprophytic growth, and pathogenicity (99). This result suggested that the fungus might be capable of compensating for the absence of the lectin by expressing other proteins with similar function(s) as the lectin in A. oligospora.

The recognition of molecular patterns associated with specific pathogens or food sources is fundamental to ecology and plays a major role in the evolution of predator-prey relationships. Recent studies have shown that nematodes could produce an evolutionarily highly conserved family of small molecules, the ascarosides, which serve essential functions in regulating nematode development and behavior. Interesting research revealed that nematophagous fungi could detect and respond to ascarosides. It was found that ascarosides, which are constitutively secreted by many species of soil-dwelling nematodes, represent a conserved molecular pattern used by nematophagous fungi to detect prey and trigger trap formation. Ascaroside-induced morphogenesis is conserved in several closely related species of nematophagous fungi and occurs only under nutrient-deprived conditions. It was demonstrated that microbial predators eavesdrop on chemical communication among their metazoan prey to regulate morphogenesis, providing a striking example of predator-prey coevolution (37).

Despite large variation in their morphology, adhesive traps share a unique ultrastructure that clearly separates them from vegetative hyphae. One common feature of traps is the presence of numerous cytosolic organelles called dense bodies (100, 101). These organelles always have catalase and d-amino acid oxidase activities, which indicate that they are peroxisome-like organelles. However, the function of these organelles is not yet fully understood. Another feature of traps is the presence of a fibrillar layer of extracellular polymers, which are believed to play an important role in the attachment of the trap cell to the nematode surface (9, 102). Following adhesion, a penetration tube forms and then pierces the nematode cuticle. At this stage, the nematode becomes paralyzed (killed), and the internal tissues are rapidly colonized by fungal hyphae (103).

After recognition and adhesion, the nematode-trapping fungi, like other pathogens, enter into the host through both enzyme degradation and mechanical pressure. The nematode cuticle is composed mainly of proteins including collagen, and several proteases have been isolated from nematophagous fungi that can hydrolyze proteins of the cuticle (104–107). Several extracellular hydrolytic enzymes including serine proteases and collagenases have been detected and partly identified from different nematode-trapping fungi. These studies suggested that extracellular hydrolytic enzymes are key virulence factors involved in the penetration process. Following penetration, the nematode is digested by the invading fungi. These fungi obtain nutrients from the nematodes for their growth and reproduction (108).

APPLICATION OF NEMATODE-TRAPPING FUNGI

In recent decades, concerns about the environmental hazards of using chemical nematicides and limited alternative crops for rotation have led to the development of biological control agents as a component of crop protection. Biological control is considered ecologically friendly and is a possible alternative in pest and disease management. Plant-parasitic nematodes cause significant damage to a broad range of vegetables and agricultural crops throughout the world. Yield losses worldwide to plant-parasitic nematodes have been estimated to range from 5 to 12% annually (3, 4, 6, 109). These losses, however, are influenced greatly by both the production systems and management options utilized. The continuous production of susceptible crops in the sandy soils of the southeastern coastal plains of the United States and other similar regions also often result in devastating losses to nematodes, especially Meloidogyne and Heterodera species (110).

As the natural enemies of nematodes, nematophagous microorganisms offer a promising approach to control the nematode pests. These biological control agents have an important effect in the regulation of plant-parasitic nematode populations, and numerous organisms including fungi, bacteria, viruses, nematodes, and other invertebrates have antagonistic activity against plant-parasitic nematodes. According to the report from MarketsandMarkets, from 2009 to the present, the market for biological nematicides has grown nearly 20%, with most of the growth in field crops and biological products occurring in the United States. Sales of nematicides totaled just over $1 billion in 2014 and are expected to grow at a compound annual growth rate of 2.7% in the next 10 years to reach $1.3 billion in 2024. The ability to trap nematodes makes nematode-trapping fungi an attractive candidate agent for controlling parasitic nematodes of plants and animals. Indeed, two commercial biological nematicides, Royal 300 and Royal 350, have been developed based on the species Arthrobotrys irregularis, A. oligospora, and Arthrobotrys robusta. Fungal biological control is an exciting and rapidly developing research area, and growing attention is being paid to the exploitation of fungi to control nematodes (3, 4, 6, 109, 111). The relationship between nematodes and the fungi that infect them has been the subject of widespread mycological studies.

Lots of natural enemies attack nematodes and decrease their populations, but the number of organisms which could be practically employed for biocontrol is restricted (112). In other words, many soil types and their microorganisms all around the world show biological control activity, but their effect on nematode populations can range from insignificant to complete suppression. There have been advances in the biological control of nematodes, but field-scale exploitation of this tactic remains to be realized. Many fungi grow slowly in natural soil, and this can be attributed to inherent competition in the highly diverse communities of soil microfauna and microflora. For a fungus to overcome the competition of resident soil microflora, it must produce a resistant resting stage that is rich in food sources (113). Also, a number of organisms, including mites and possibly mycophagous nematodes, may feed on mycelium of these fungi. Some disadvantages such as complexity in the establishment in the soil, their limited capturing activity, and above all, nonspecific trapping of plant-parasitic nematodes reduce their potential for biological control. Some Arthrobotrys species have been formulated and applied under specific conditions, but the results were inconsistent. So there is still a long way to go in developing a highly efficient fungal agent to control nematode pests.

GENOMICS AND PROTEOMICS OF NEMATODE-TRAPPING FUNGI

Genomic and proteomic analyses provided comprehensive understanding of the evolution and infection process of fungi. Recently, the evolution of the nematode-trapping lifestyle and the infection process, including the differentiation of trap cells and the penetration and digestion of nematodes, have been examined using genomic and proteomic approaches.

So far, several genomes of nematode-trapping fungi have been de novo sequenced or are currently being sequenced, including Arthrobotrys conoides, A. oligospora, Dactylellina appendiculata, Dactylellina drechsleri, Dactylellina haptotyla, D. stenobrocha, Gamsylella cionopaga, Gamsylella querci, and Orbilia auricolor. Genomics or proteomics analyses of A. oligospora forming adhesive networks (36), Monacrosporium haptotylum forming adhesive knobs (35), and D. stenobrocha forming constricting rings (33) have been published and provided a new approach for obtaining a better understanding of the interaction between nematode-trapping fungi and their hosts.

Molecular phylogenies based on rDNA sequences strongly support that the nematode-trapping lifestyle evolved once in the Orbiliales. Subsequently, several distinct trapping types evolved. On the genomic level there are three compatible mechanisms that could account for such evolutionary patterns. First, parasitism has been associated with the presence of novel genes. Such genes could be acquired by gene duplication or horizontal gene transfer. Second, adaptations to the parasitic habit may result from the differences in the regulation of gene expression. Third, parasitism is associated with gene loss and deletions (114). By comparative genomics analysis of the knob-forming species M. haptotylum and the net-forming species A. oligospora, two genomic mechanisms have been determined to be important for the parasitic adaptation in these fungi. First, the expansion of protein domain families and the large number of species-specific genes indicated that gene duplication followed by functional diversification had a major role in the evolution of the nematode-trapping fungi. Gene expression indicated that many of these genes are related to pathogenicity. Second, the gene expression of orthologs between the two fungi during the infection process indicated that differential regulation was an important mechanism for the parasitic evolution in nematode-trapping fungi (35).

Comparative genomics analysis showed that A. oligospora shared many more genes with pathogenic fungi than with nonpathogenic fungi. Specifically, compared to several sequenced ascomycetous fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR analyses revealed that 90 genes were significantly upregulated at the early stage of trap formation by nematode extracts, and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, and cell wall and membrane biogenesis. Based on the combined genomic, proteomic, and qPCR data, a model was proposed for understanding the formation of the nematode-trapping device in this fungus. In the model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation, and peroxisome biogenesis (35, 36).

The trap of M. haptotylum consists of a unicellular structure called a knob that develops at the apex of a hypha. To better understand the cellular functions of adhesive traps, the trap cell proteome of the fungus M. haptotylum was characterized. In this research, 336 proteins were identified, with 54 expressed at significantly higher levels in the knobs than in the mycelia. The upregulated knob proteins included peptidases, small secreted proteins with unknown functions, and putative cell surface adhesins containing carbohydrate-binding domains, including the WSC (cell wall integrity and stress-response component) domain. A further phylogenetic analysis showed that all upregulated WSC domain proteins belonged to a large, expanded cluster of paralogs in M. haptotylum. Several peptidases and homologs of experimentally verified proteins in other pathogenic fungi were also upregulated in the knob proteome. Complementary profiling of gene expression at the transcriptome level showed poor correlation between the upregulation of knob proteins and their corresponding transcripts. It was concluded that the traps of M. haptotylum contain many of the proteins needed in the early stages of infection and that the trap cells can tightly control the translation and degradation of these proteins to minimize the cost of protein synthesis (115).

These genomic and proteomic studies will greatly facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungal-nematode interactions.