Fungus Nematode Interactions

5.1.1 Nematode-Trapping Fungi

Arthrobotrys oligospora forms so-called adhesive network traps on which vermiform nematodes are captured. The formation of traps in this fungus is induced chemically by

Figure 3 Top figures show infection of nematode egg by the egg parasite Pochonia sp. Germling of the fungus forms an appressorium after contact with the egg surface (left). An adhesive is formed and the fungus penetrates the egg shell, grows inside the egg, and digests its contents. Bottom figures illustrates capture and infection of a nematode by the nematode-trapping fungus Arthrobotrys sp. A nematode is captured in the three-dimensional network trap (left). The middle figure (enlargement of left figure) shows the trap, covered with adhesive, penetration of the nematode cuticle, and formation of an infection bulb. The trap and infection bulb contain dense bodies (dark dots). The right figure shows an enlargement of the middle figure with the multilayered nematode cuticle covered with a surface coat.

Figure 3 Top figures show infection of nematode egg by the egg parasite Pochonia sp. Germling of the fungus forms an appressorium after contact with the egg surface (left). An adhesive is formed and the fungus penetrates the egg shell, grows inside the egg, and digests its contents. Bottom figures illustrates capture and infection of a nematode by the nematode-trapping fungus Arthrobotrys sp. A nematode is captured in the three-dimensional network trap (left). The middle figure (enlargement of left figure) shows the trap, covered with adhesive, penetration of the nematode cuticle, and formation of an infection bulb. The trap and infection bulb contain dense bodies (dark dots). The right figure shows an enlargement of the middle figure with the multilayered nematode cuticle covered with a surface coat.

small peptides, e.g., phenylalanyl valine (Nordbring-Hertz 1973) or by nematodes (Nordbring-Hertz 1977). The presence of traps is a prerequisite for infection of living nematodes, and in fact increases the ability of the fungus to chemically attract nematodes (Jansson 1982). After contact between the fungal trap and the nematode cuticle (Figure 3 bottom left) a possible contact recognition step occurs involving a fungal lectin binding to N-acetylgalactose amine (Gal-NAc) on the nematode surface (Nordbring-Hertz and Mattiasson 1979). The nematode surface, the cuticle (Figure 3 bottom right), consists of several layers containing proteins (mainly collagen), lipids, and carbohydrates (Bird and Bird 1991). Externally to the cuticle a surface coat (or glycocalyx) consisting of glycoproteins is found (Bird and Bird 1991). The surface coat is probably the part of the nematode surface most relevant to recognition and adhesion of nematophagous fungi, since proteolytic removal of this structure results in reduced adhesion of bacteria and spores of endoparasitic nematophagous fungi (Bird 1985; Jansson 1993).

The trapping organ of A. oligospora contains an adhesive material. Upon contact with the nematode surface, a recognition step possibly mediated, at least partly, by lectin binding, induces changes in the structure of the adhesive leading to capture of the nematode (Veenhuis et al. 1985). The adhesive undergoes changes from an amorphous material to a fibrillar structure, more organized with fibrils perpendicular to the nematode surface. This may anchor the nematode to the trap thus facilitating infection. In contrast, the adhesive of the endoparasitic fungus D. coniospora does not appear to change and has a fibrillar structure even in the absence of nematodes (Jansson and Nordbring-Hertz 1988). After the firm attachment to the host surface, A. oligospora penetrates the nematode cuticle and forms an infection bulb (Figure 3 bottom middle), from which trophic hyphae grow out to digest the nematode contents (Veenhuis et al. 1985). In the trap and the infection bulb organelles, so-called dense bodies, now appear. These organelles are not present in ordinary hyphae and have been suggested to contain hydrolytic enzymes used for penetration of the cuticle and digestion of host (Jansson and Nordbring-Hertz 1988; Veenhuis et al. 1985).

As in many other instances of fungal penetration of their hosts' surfaces, nematophagous fungi appear to use both enzymatic and physical means. The nematode cuticle mainly

Biological Control Nematodes

Figure 4 Hypothesis of interference with nematode chemotaxis. Conidia of the endoparasitic nematophagous fungus Drechmeria coniospora adhere to the sensory organs at the anterior end of a nematode and block nematode attraction (left). The top right figure illustrates the tip of the nematode head with two amphids (chemosensory organs). The chemotactic factors (black dots) are transported to the neuron membranes where the chemoreceptors are located. (A) shows a proteinaceous chemoreceptor with carbohydrate chains, where the terminal sugar (purportedly mannose or sialic acid, triangle) binds to a chemotactic factor leading to normal attraction of the nematatode. (B) Illustrates blocking of terminal carbohydrates with lectins (Con A and Limulin) thereby inhibiting nematode chemotaxis. (C) Shows enzyme (mannosidae and sialidase) obliteration of terminal sugar moieties leading to inhibition of chemotactic behavior. Proteolytic enzymes, hydrolyzing the chemoreceptor (membrane protein), have a similar effect.

Figure 4 Hypothesis of interference with nematode chemotaxis. Conidia of the endoparasitic nematophagous fungus Drechmeria coniospora adhere to the sensory organs at the anterior end of a nematode and block nematode attraction (left). The top right figure illustrates the tip of the nematode head with two amphids (chemosensory organs). The chemotactic factors (black dots) are transported to the neuron membranes where the chemoreceptors are located. (A) shows a proteinaceous chemoreceptor with carbohydrate chains, where the terminal sugar (purportedly mannose or sialic acid, triangle) binds to a chemotactic factor leading to normal attraction of the nematatode. (B) Illustrates blocking of terminal carbohydrates with lectins (Con A and Limulin) thereby inhibiting nematode chemotaxis. (C) Shows enzyme (mannosidae and sialidase) obliteration of terminal sugar moieties leading to inhibition of chemotactic behavior. Proteolytic enzymes, hydrolyzing the chemoreceptor (membrane protein), have a similar effect.

contains proteins (Bird and Bird 1991) and therefore the action of proteolytic enzymes may be important for penetration. A serine protease, PII, from A. oligospora, has been characterized, cloned, and sequenced (Ahman et al.

1996). The expression of PII is increased by the presence of proteins, including nematode cuticles (Ahman et al. 1996). PII belongs to the subtilisin family and has a molecular mass of 32kDa (for review see Jansson et al. 1997). Serine proteases will also be discussed in "Egg Parasites." During decomposition of infected nematodes A. oligospora produces a lectin (A. oligospora lectin, AOL), which functions as a storage protein within the nematode host, and may constitute as much as 50% of the total fungal protein. AOL is a multispecific lectin, which binds to sugar chains common in animal glycoproteins, including those of nematodes (Rosen et al.

5.1.2 Egg Parasites

Egg-destroying fungi act on nematode eggs at two levels: directly as true parasites by penetrating and infecting eggs, and indirectly by causing distortions in the larvae or embryos they contain (Morgan-Jones and Rodrigues-Kabana 1988). The former mode of action is well documented and is largely responsible for cases of soil supressiveness to nematodes. In this chapter we will describe the direct mode of fungal infection of nematode eggs mainly using the egg-parasitic fungi Pochonia rubescens (syn. Verticillium suchlasporium) and P. chlamydosporia (syn. V. chlamydosporium).

Upon growth of germ tubes the hyphal tips swell and differentiate into appressoria at contact with nematode eggs (Lopez-Llorca and Claugher 1990) (Figures 2 and 4), as well as on artificial, especially hydrophobic surfaces (Lopez-Llorca et al. 2002b). An extracellular material (ECM) probably functions as adhesive, but possibly also seals the hole caused by the penetration hypha (Figure 3, top). This ECM can be labeled with the lectin Concanavalin A, indicating that the ECM contains mannose/glucose moieties probably on side chains of glycoproteins (Lopez-Llorca et al. 2002b). Most ECMs of fungal hyphae consist of proteins and carbohydrates (Nicholson 1996).

Early evidence of eggshell penetration by P. chlamydosporia came from studies of plant- and animal-parasitic nematode egg infection (Lysek and Krajci 1987; Morgan-Jones et al. 1983). In eggshells, low electron dense areas were found in the vicinity of penetration hyphae of P. rubescens on cyst nematodes (Lopez-Llorca and Robertson 1992a) suggesting involvement of eggshell degradation enzymes. Nematode eggshells mostly contain protein and chitin (Clarke et al. 1967) organized in a microfibrillar and amorphous structure (Wharton 1980). Therefore, a search for extracellular enzymes degrading those polymers was carried out. A 32-kDa serine protease (P32) was first purified and characterized from the egg parasite P. rubescens (Lopez-Llorca 1990). Involvement of the enzyme in pathogenesis was suggested by quick in vitro degradation of Globodera pallida egg shell proteins (Lopez-Llorca 1990), but most of all by its immunolocalization in appressoria of the fungus infecting Heterodera schachtii eggs (Lopez-Llorca and Robertson 1992b). Although pathogenesis is a complex process involving many factors, inhibition of P32 with chemicals and polyclonal antibodies reduced egg infection and penetration (Lopez-Llorca et al. 2002b). The similar species P. chlamydosporia also produces extracellular proteases (VcP1) (Segers et al. 1994), which are immunologically related to P32 and similar enzymes from entomopathogenic fungi (Segers et al. 1995). VcP1-treated eggs were more infected than untreated eggs suggesting a role of the enzyme in eggshell penetration by egg-parasitic fungi. Recently, several chitinolytic enzymes of Pochonia spp. were detected. One of those accounting for most of the activity was a 43-kDa endochitinase (CHI43) (Tikhonov et al. 2002). When G. pallida eggs were treated with both P32 and CHI43, damage to eggshell was more extensive than with each enzyme alone, suggesting a co-operative effect of both enzymes to degrade egg shells (Tikhonov et al. 2002).

5.1.3 Interference with Nematode Chemoreception

Carbohydrates present on nematodes are not only involved in the recognition step of lectin binding, but also appear to be involved in nematode chemotaxis (Jansson 1987; Zuckerman and Jansson 1984). The main nematode sensory organs are the amphids and the inner labial papillae located around the mouth in the head region of the nematodes (Ward et al. 1975). Figure 4 (right) schematically depicts the structure of amphids in the nematode head. The chemoreceptors are thought to be located on the neuron membrane. There are 28 neurons in the nematode Caenorhabditis elegans, each with a passage to the environment, but the number of receptors is not known (Ward 1978).

A hypothesis of the involvement of carbohydrates in nematode chemoreception was put forward by Zuckerman (1983); Zuckerman and Jansson (1984). The chemoreceptors, purportedly glycoproteins, could be blocked (Figure 4B) by lectins (Concanavalin A binding to mannose/glucose residues, and Limulin binding to sialic acid) resulting in loss of chemotactic behavior of bacterial-feeding nematodes to bacterial exudates (Jeyaprakash et al. 1985). Furthermore, treatment of the nematodes with enzymes (mannosidase and sialidase) thus obliterating the terminal carbohydrates (Figure 4C) also resulted in decreased chemotactic behavior (Jansson et al. 1984), demonstrating the importance of these carbohydrate moieties in nematode chemotaxis.

Interfering with nematode chemotaxis, thereby inhibiting their host-finding behavior, may be a possible way of controlling plant-parasitic nematodes. In a pot experiment using tomato as host plant and Meloidogyne incognita as parasitic nematode, addition of Concanavalin A and Limax flavus agglutinin (sialic acid specific lectin) resulted in decreased plant damage by the nematode compared to controls (Marban-Mendoza et al. 1987). Addition of lectins (or enzymes) on a field is not feasible, but the possibility to use, for instance, lectin-producing leguminous plants have

been shown to reduce galling by root-knot nematodes (Marban-Mendoza et al. 1992).

The endoparasitic nematophagous fungus D. coniospora infects nematodes with its conidia, which adhere to the chemosensory organs (Figures 2 and 4) (Jansson and Nordbring-Hertz 1983). Conidial adhesion was suggested to involve a sialic acid-like carbohydrate since treatment of nematodes with the lectin Limulin, and treatment of the spores with sialic acid, decreased adhesion (Jansson and Nordbring-Hertz 1984). Furthermore, nematodes with newly adhered spores lost their ability to respond chemotactically to all attraction sources tested, i.e., conidia, hyphae, and bacteria, indicating a connection between adhesion and chemotaxis through carbohydrates on the nematode surface (Jansson and Nordbring-Hertz 1983). The conidia of D. coniospora adhere to the chemosensory organs of Meloidogyne spp., but do not penetrate and infect the nematodes. Irrespective of the lack of infection the fungus was capable of reducing root galling in tomato in a biocontrol experiment (Jansson et al. 1985), again indicating the involvement of chemotactic interference.

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