Keywords

1 Introduction

Biological control is a subject that covers a huge area of application, bugs to control other bugs for application in medical, animal husbandry and agricultural situations. Stirling (1991) defined biological control of plant- parasitic nematodes as, ‘A reduction of nematode populations which is accomplished through the action of living organisms other than nematode-resistant plants, which occurs naturally or through the manipulation of the environment or the introduction of antagonists’. In all these situations the underlying biology rests on the generic science pertaining to an interaction of one organism with another organism, either directly or indirectly, and it is sufficiently well understood that it can be used and be exploited to reduce an unwanted organism, in this case a nematode pest, to a number whereby it no longer has a detrimental effect on crop yield.

1.1 Historical Context

Prior to the use of nematicides, biological control was always viewed as a possible way to control plant-parasitic nematodes and as early as 1920 Cobb had suggested the use of predatory nematodes in sugar beet fields to control Heterodera schachtii (Cobb 1920). Further research suggested this approach was not economically viable (Thorne 1927). Several years later the focus changed to the use of predatory fungi and in particular the use of nematode trapping fungi (Linford 1937; Linford et al. 1938). However, these promising results were eclipsed by the spectacular results that began to be obtained by the newly developed nematicides (Stirling 1991). Throughout the later part of the twentieth century plant-parasitic nematodes have successfully been controlled by the use of these compounds, which were manufactured through the petro-chemical industry. More recently, with the increasing recognition of their toxicity and their harmful effects to both the environment and human health, legislation has been implemented to reduce their use. Although biological control has always tended to be in the shadow of plant resistance as an alternative to chemical control, it is again back on the agenda.

The focus of this renewed research effort into biological control was built on the observation that although mono-cropping can lead to an increase in plant-parasitic nematodes, with a concomitant reduction in crop yield, continued mono-cropping leads to the production of nematode suppressive soils (Gair et al. 1969). Subsequent research has shown that predatory fungi were responsible for this suppression (Kerry 1975; Kerry and Crump 1977; Kerry et al. 1982). The recognition that parasites were responsible for the development of nematode suppressive soils lead to the ‘classical’ approach to biological control where parasites were identified in nematode suppressive soils, mass produced and introduced into areas were nematode pests were abundant (Stirling 1991). Despite the best efforts of biological control scientists over the last 20–30 years, and although impressive results have been obtained using this approach, generally speaking this has not produced a robust method of nematode control which is consistent and broadly applicable. To date, there are relatively few commercially available products (Whipps and Davies 2000; Hallmann et al. 2009).

1.2 Technical Developments and Twenty-First Century Science

During the last decades of the twentieth century most research groups focused on attempting to isolate soil microorganisms, mainly fungi and bacteria, without devoting any special concern as to the mode-of-action of these potential microbial control agents. Early research that explored the mechanistic interactions between plant-parasitic nematodes and trapping fungi initially started out as fundamentally morphological in nature. This research later developed to examine biochemical aspects of infection processes and this approach continues today with an emphasis on molecular and genomic aspects (Tunlid and Ahrén 2010; Davies and Spiegel 2010). Similar trends can be seen within other research groups and this emphasises the interaction between the growth of biological control knowledge through research and its dependent relationship with technological development. For example, initial morphological studies exploited the use of light- and electron microscopy, which in turn expanded into investigations on ‘ecological’ aspects based on observations made using microbiological techniques such as selective media for the quantification of microbial populations in soil (Kerry and Hirsch 2010). The first decade of the twenty-first Century witnessed the use of a combination of approaches (molecular biology, biochemistry, genetics/genomic and developmental biology) aimed at getting a deeper understanding of such interactions (Davies and Spiegel 2010) and the challenge is therefore to bring a coherence to these approaches in an effort to develop robust control strategies that can work in the field situation.

2 Ecological Context

The most economically important plant-parasitic nematodes are root parasites of crops, where they inhabit the water films on soil particles and can feed on plant roots. This environment is highly fragmented and subject to continual wetting and drying. Because nematode movement is only possible between a water tension of 4.2 (a level at which plants are wilting) and 4.45 pF (Jones and Jones 1974), the hydraulic properties of the soil are critical and these are continually changing both spatially and temporally (Avendano et al. 2004).

2.1 The Soil as a Heterogeneous Environment

As large amounts of carbon are exuded by plant roots (Bekku et al. 1997) this carbon source is an important driver for determining the population dynamics of organisms occurring in the rhizosphere where nematodes reside as part of the community. Roots that permeate the soil are not homogeneous in their distribution but exploit nutrient rich patches which lead to changes in root morphology (Hodge 2006; Watt et al. 2006). Plant-parasitic nematodes, which are sensitive to the changing chemical properties of the rhizosphere, use these to locate plant host roots and therefore they are also patchily distributed (Costa et al. 2010).

2.2 Food-Webs and Multitrophic Interactions

Plant-parasitic nematodes form a part of a below-ground tritrophic interaction between plant roots and the soil/root microbial community in which the populations of phytonematodes are controlled by a number of different processes that include bottom up, horizontal, and top down control.

2.2.1 Bottom up Control

Bottom up control is where a population of nematodes is determined by the level of nutrients available. In the case of plant parasitic nematodes, which are obligate parasites of plants, this has involved a co-evolving arms race between host plants and parasitic nematodes. It is interesting that different species of plant-parasitic nematodes have adopted different life-cycle strategies that arguably form an evolutionary continuum from migratory ectoparasites, through to migratory endoparasites and on to sedentary endoparasites (Wood 1973; Yeates et al. 1993) exhibiting increasing specialisation. Interestingly not all plants are equally susceptible to nematode parasitism and different plants exhibit differing degrees of resistance and/or tolerance (Cook and Starr 2006; Trudgill 1991) and of the most economically important nematodes, the cyst nematodes are regarded as considerably more host specific than the polyphagous root-knot nematodes. However, due care needs to be taken when investigating such parameters because the susceptibility of a particular plant to a particular nematode may be due to endophic fungi and or arbuscular mycorrhizal fungi (Hallmann and Sikora 2010) that may be playing an important role in bottom up control. The role of rhizobia on legumes and their ability to reduce nematode populations is much less clear with reports of some strains of rhizobia capable of inducing resistance in the root against plant-parasitic nematodes (Reitz et al. 2000; Mitra et al. 2004).

2.2.2 Horizontal Control

The maximum number of nematodes that can be sustained on a given root system represents its carrying capacity and the population increase can be modelled as a curve in which there is increased intra-specific competition for the available niches present on the root as the number of individual nematodes increases (Varley et al. 1973). Two species of nematode that reproduce continually and have overlapping ecological niches would also be subjected to intra-specific competition at low population densities. However, as populations of each species became larger they would increasingly be subjected to inter-specific competition, the outcome of which would be that one nematode species would be reducing the number of the other species (McSorley and Duncan 2004; Van den Berg and Rossing 2005). This control of one species of nematode by another represents horizontal control (Van der Putten et al. 2001). Competition experiments have been undertaken to evaluate the extent to which horizontal control plays a role in determining the population densities of Meloidogyne maritima, Heterodera arenaria and Pratylenchus penetrans in marram grass on costal sand dunes and it appeared that the outcomes were mediated by the host plant (Brinkman et al. 2005; de la Pena et al. 2008).

2.2.3 Top Down Control

The use of top down control is the strategy employed by most scientists envisaging the use of inundation of a selected natural enemy, usually a fungus or a bacterium, to control a particular plant-parasitic nematode. This method is based on the isolation of a nematode natural enemy that is parasitic on a particular nematode pest from a field that is nematode suppressive, mass produced and then deployed back to a field where the nematode is the cause of yield decline. Although this method has proved useful in pot tests and glasshouse studies, scaling up this approach to the field scale has been problematic leading to variable results. Analysis of the problem from an ecological perspective may therefore provide useful insights.

Field populations of nematodes usually occur as mixtures, both mixtures of different species and mixtures of different sub-species. Therefore in a nematode suppressive soil one would imagine that the natural microbial enemies of such a community would also contain a mixture of different species of microbial enemies and sub-species of microbial enemies. What follows a priori from the Red Queen Hypothesis (Van Valen 1973, 1976) suggests that a co-evolutionary arms race between hosts and parasites will lead to genetic diversity in which hosts are driven to produce genetic combinations which decrease parasite virulence, whereas parasites are driven to produce genetic combinations to maintain virulence. The results of such an arms race over extended time would be to allow infection and multiplication of the parasite without a detrimental effect on the host (Ebert and Hamilton 1996).

Co-evolutionary mathematical models predict that parasites that have a broad host range will tend to diverge, generating sub-populations with different host specializations. Because of the smaller size and quicker generation times of microbial parasites over their hosts each microbial subpopulation will co-evolve faster with its host, than a generalist population can evolve with different and variable hosts (Kawecki 1998). Therefore, at the initiation of such a host-parasitic arms race, it is not surprising that boom and bust population dynamics are the norm, and that the success of any selected microbial enemy from a particular pest nematode, deployed back into a field will produce inconsistent results. Investigations looking at microbial diversity and parasitism seem to support this view (see Sect. 23.4).

3 Molecular Approaches for Assessing Field Biodiversity

To gain insights into nematode suppression for the development of biological control strategies it is becoming increasingly obvious that knowledge of the diversity of the nematodes and their natural enemies, together with their temporal and spatial distribution, will play a key role. Conventional methods of identifying and quantifying both nematodes and their microbial enemies is time consuming and labour intensive and requires a high level of technical expertise, which, when all taken together, compromises the number of samples that can be processed (Costa et al. 2010). In addition, because of the heterogeneous spatial distribution of nematodes and their microbial enemies, and the limitations of soil sampling, most sampling techniques usually average the distribution of organisms and vary in their efficiency of their extraction (Ettema and Wardle 2002; McSorley and Frederick 2004). The population dynamics of host-parasite interactions occurring in the soil are therefore notoriously difficult to ascertain. The ability to quantify relationships between hosts and their parasites will be important in the development of biological control strategies and new molecular approaches have an important role to play.

3.1 Antibodies

Antibodies are a class of globular proteins produced by the lymphatic system of animals in response to its invasion by a foreign compound or organism. The function of antibodies is to label these foreign bodies for destruction by the immune system. Antibody binding is very specific and this specificity can be used as a tool in the identification and diagnosis of pests and other organisms (Clark 1994). Antibodies have been shown to be useful in the identification and quantification of both plant-parasitic nematodes and their microbial enemies (Davies 1994). Antibodies can be made with variable degrees of specificity and theoretically can be made into a diagnostic tool for any level of molecular operational taxonomic unit (MOTU) required. Once produced, they are generally easy to use in a number of different formats from dip stick type assays and enzyme linked immune-assays through to fluorescence microscopy (Clark 1994). They can be used qualitatively as well as quantitatively and are therefore highly flexible (Davies et al. 1996). Monoclonal antibodies have been used to differentiate species of both root-knot nematodes (Davies and Lander 1992) and potato cyst nematode (Robinson et al. 1993) and can be used to quantify nematodes in soil samples (Davies and Carter 1995). Antibodies have also been successful in monitoring the nematophagus fungus Pochonia chlamydosporia and the bacterial parasite Pasteuria penetrans in the rhizosphere using immunofluorescence microscopy (Hirsch et al. 2001; Costa et al. 2006).

3.2 Phospholipid Fatty Acid Analysis (PFLA)

Fatty acids can be used in the classification and identification of microorganisms (O’Donnell 1994) and the interaction of nematodes with their fungal and bacterial communities has been studied using PFLA profiles (Bardgett et al. 1996; Denton et al. 1999). Changes in PLFA profiles reflect changes in the microbial community structure and are indicative of the biomass of the various groups of bacteria and fungi present (Bardgett et al. 1996). However, although the method is sensitive enough to monitor changes to some different bacterial groups, all fungi are measured by one particular fatty acid and this therefore limits their application (Denton et al. 1999). Thus, although PFLAs have been useful in monitoring changes in the microbial communities in relationship to environmental conditions (O’Donnell et al. 2005), they have limited use in assessing microbial diversity in any detail. However, PFLAs have been influential in studying the interactions and inter-connectedness of different trophic groups of nematode populations in a below ground grassland ecosystem (Bardgett et al. 1999).

3.3 DNA

Identification of organisms using DNA-based techniques on a routine basis dates back to the 1980s but was revolutionised by the invention of the Polymerase Chain Reaction (PCR) which relies on enzymatic amplification of a given DNA sequence (Saiki et al. 1985). A PCR-based method called denaturing gradient gel electrophoresis (DGGE) has enabled community fingerprints of large groups of microorganisms to be obtained that provides a measure of their genetic diversity (Muyzer et al. 1993). Originally this technique was applied to bacterial communities in soil by amplifying the 16s rDNA gene and this combined with the sequencing of excised bands from the original gel can provide further taxonomic information (De Mesel et al. 2004). Similar approaches, using 18s rDNA have been applied to fungal communities but their quantification is complicated by the inability of the method to distinguish fungal spores from vegetatively growing hyphal fragments (van Elsas et al. 2000). DGGE analysis has been used to show that plant defence against nematodes was not mediated by changes in the soil microbial community (Wurst et al. 2009). Similar approaches have also been used to study nematode communities (Waite et al. 2003), but, because the number of MOTUs that can be used for describing nematodes is still very small the technique is currently only of limited value (Foucher et al. 2004; Wu et al. 2009).

Another PCR-based approach for characterising nematode communities has been the use of molecular barcodes using the 18s rDNA subunit which are designed to relate to MOTUs (Floyd et al. 2002; Blaxter et al. 2005). Systems are being constructed where it will be possible to use molecular barcodes to obtain sequences that can be used to interrogate online databases to obtain nematode identifications (Powers 2004). Indeed, using these approaches it has been possible not only to identify nematodes but also to construct phylum-wide evolutionary trees (Blaxter et al. 1998; Holterman et al. 2006) that recently have shed light on the evolution of parasitism amongst plant parasitic nematodes (Holterman et al. 2009). Taking these approaches forward it should be possible to elucidate and help understand nematode parasitic interactions such that light will be shed on the functional interactions between the organisms within the rhizosphere community.

3.4 Ecological Genomics

Studies of nematode control in their ecological context, particularly top down control which will probably give the most insights into organisms with biological control potential, will need all these molecular approaches to understand the necessary population dynamics that will underpin the development of nematode biological control strategies. The challenge for the biological control scientist is to integrate research in such a way that a model of the rhizosphere community can be developed. From the perspective of controlling plant -parasitic nematodes the new genome sequences (Abad et al. 2008; Opperman et al. 2008), and very shortly those of other nematode species, will be invaluable. This, together with the sequencing of both their plant hosts, and followed by the sequencing of their parasites, will allow the possibility of a multitrophic ecological genomic approach. Rhizospheric ecological studies will then allow the integration of different levels of organisation in which changes in the genetic makeup of a host with its parasites at the population level will be integrated with the specific mechanistic host-parasite interactions at the biochemical and molecular level (Zheng and Dicke 2008).

4 Towards Understanding Field Variation Through Molecular Mechanisms: Three Models

If the knowledge gained from sequencing genomes is to be integrated into understanding the population genetics of host-parasitic interactions, the identification of the functional genes involved in compatible and incompatible host—parasite interactions at the molecular level becomes indispensible. In microbial populations, as discussed above, such genes are likely to be involved in arms races that conform to the Red Queen Hypothesis which would lead to the prediction that these genes will have diversified (Van Valen 1973, 1976). Within the context of the soil environment, which we know is likely to be highly heterogeneous with respect to both hosts and parasites, this produces the challenge of species designation or MOTU identification within the context of the metapopulation structure (Fraser et al. 2009) present in the soil habitat. Therefore, understanding the functional mechanisms will be a necessary key to structuring the host-parasite metapopulations in soil and how these interact. This knowledge will be important for the applied biological control scientist to determine in which situations biological control as a management strategy will be successful. Three models are used to explore functional mechanisms of the interactions between potential biological control agents and plant-parasitic nematodes.

4.1 Arthrobotrys

Fungi have long been known to be parasitic on nematodes (Barron 1977) and more than 200 species of nematophagous fungi have been described (Tunlid and Ahrén 2010). One group of these are the nematode trapping fungi which, under conditions of limited nutrient availability and in the presence of nematodes, produce specialised organs that can capture nematodes (Barron 1977). The trapping of nematodes by fungi can be broken up into three distinct stages, starting with trap production, followed by recognition and capture and finally penetration and digestion. These three stages overlap to some extent and are not totally separate.

Many trapping fungi produce traps spontaneously, but as early as the 1950s it was shown that “nemin”, a mixture of compounds produced by the nematode, stimulated the formation of traps in A. conoides (Pramer and Stoll 1959; Pramer and Kuyama 1963). One particular group of trapping fungi, Arthrobotrys spp., have been studied in detail and microscopic studies of A. oligospora have shown that the production of traps and subsequent infection of nematodes occurs in a sequence of events, as outlined above, that is completed within 48–60 hours (Dijksterhuis et al. 1994). When a nematode makes contact with a trap a layer of extracellular fibrils that surround the traps become directed perpendicularly to the nematode cuticle and the fungus produces a penetration tube that is thought to be involved in production of hydrolytic enzymes that help to solubilise the cuticle. Then the nematode becomes paralysed and the penetration tube forms an infection bulb from which hyphae develop and colonise the captured nematode (Tunlid and Ahrén 2010).

4.1.1 Lectin-Carbohydrate Interactions

Lectins are proteins that bind carbohydrates and function as recognition molecules (Sharon and Lis 2004). Early experiments based on sugar inhibition and red blood cell (RBC) assays suggested that a carbohydrate present on the surface of the nematode cuticle was interacting with an N-acetygalactosamine (GalNac)-specific lectin present on the surface of the traps in A. oligospora (Nordbring-Hertz and Mattiasson 1979). This specificity was confirmed with the finding that Type A RBCs, which contain a terminal GalNac, bound more easily than did Type B and O (Borrebaeck et al. 1984; Premachandran and Pramer 1984). However, the situation is complex, with different species of fungi revealing different binding specificities (Nordbring-Hertz and Chet 1988). Purification of these lectins (Rosén et al. 1992), and more recently the development of specific deletion mutants to see if they played and important role in nematode infection processes (Tunlid et al. 1999; Balogh et al. 2003), suggests they are part of a growing family of lectins that are specific to fungi (Tunlid and Ahrén 2010).

4.1.2 Caenorhabdits elegans, Genomics and Innate Immunity

Caenorhabditis elegans is a free-living nematode that has been used as a model organism for studying aspects of developmental biology and was the first multicellular animal to be sequenced. Caenorhabditis elegans is currently being used to study aspects of pathogenesis and innate immunity (Millet and Ewbank 2004; Darby 2005; Gravato-Nobre and Hodgkin 2005; Fuchs and Mylonakis 2006). The production of strains of C. elegans with cuticles that have different surface properties makes it particularly amenable as an experimental tool for dissecting the interactions between the cuticle and microbial pathogens. Many bacteria and fungi have been shown to be pathogens of C. elegans (Hodgkin and Partridge 2008). Arthrobotrys spp. are regarded as predators of C. elegans and bioassays have shown that these nematode trapping fungi differ in their ability to trap mutants of C. elegans that show altered lectin staining to the cuticle surface (Mendoza et al. 1999).

More recently, in a study of the infection of C. elegans with Monacrosporium haptotylum, the most highly up-regulated fungal gene was a subtilisin that was similar in sequence and expression profile to PII of A. oligospora and designated spr1 (Fekete et al. 2008). However, another subtilisin spr2, although up-regulated at one hour, was then subsequently down- regulated at four and 16 hours only to be up-regulated again at 24 hours when the fungus was digesting the killed nematode (Tunlid and Ahrén 2010). Clearly, although these two genes were from the same family of subtilisins, their expression profiles would suggest they have very different functions.

Upon infection, C. elegans responds by the activation of several intracellular signalling pathways which led to the expression of defensive gene products or effector molecules (Millet and Ewbank 2004; Gravato-Nobre and Hodgkin 2005). There are several distinct signal transduction cascades including DAF2/DAF16, MAP kinase and the TGF-β pathway as well as several others none of which appear totally independent but form an integrated signalling network. However, analysis of the expression profiles of the worm showed no distinct shifts which could be detected between one and four hours after infection (Fekete et al. 2008; Tunlid and Ahrén 2010). The most responsive up-regulated gene in this study was dod-3 which is regulated by the transcription factor DAF-16. Other genes that responded to M. haptotylum infection included cnc-4 that codes for one of eleven caenacin peptides that are secreted in response to Drechmeria coniospora infection, and lec-8 and lec-10 that encode galactose binding proteins (Fekete et al. 2008) which are also up-regulated during bacterial infection (Mallo et al. 2002; Gravato-Nobre and Hodgkin 2005). Interestingly, C-type lectins which have also been implicated in the response of C. elegans to infection (O’Rourke et al. 2006) were all down-regulated.

4.2 Trichoderma

Trichoderma spp. are a group of free-living fungi that are common in the rhizosphere. They can colonise root-surfaces and even establish themselves in the root epidemis. Roots colonized by Trichoderma grow better and confer resistance to abiotic (Yedidia et al. 2001, 2003; Harman et al. 2004) and biotic stresses (Herrera-Estrella and Chet 1998; Harman 2006). Various mechanisms have been proposed for their biological control activity against root pathogens which include both direct interactions such as parasitism, antibiosis and competition and indirect mechanisms such as plant systemic induced resistance (Harman et al. 2004, 2006; Viterbo et al. 2007). It is thought that in situations where biological control occurs it is a result of a multi-mechanism action (Sharon et al. 2010). Trichoderma spp. have been tested for their ability to control plant- parasitic nematodes (Windham et al. 1989; Reddy et al. 1996; Khan and Saxena 1997; Rao et al. 1998; Meyer et al. 2000, 2001; Sharon et al. 2001; Spiegel et al. 2007) but with mixed success. Clearly, if multiple modes of action are involved, understanding the importance of each of these, from attachment and infection processes through to antibiosis, will help in developing control agents with improved potential.

4.2.1 Conidial Attachment

Conidial attachment to the nematode surface is a key step in the infection process and appears to show a degree of specificity as conidia will attach to gelatinous egg matrix (gm) and nematodes that had had contact with the gm but not with gm-free J2s and eggs (Sharon et al. 2007). Application of antibodies to the surface coat of infective juveniles increased attachment of conidia to the nematodes. This increase in parasitism possibly occurs through the process of bilateral binding in which the antibody forms a bridge between the conidium and the juvenile and thereby increases the rates of infection (Sharon et al. 2010). Further experiments have suggested that a Ca2+-dependent lectin-carbohydrate interaction may be involved (Sharon et al. 2007, 2009).

4.2.2 Lytic Enzymes

Trichoderma spp. produce a whole array of different enzymes that are involved in plant defense and in biological control processes (Viterbo et al. 2002a, b; Markovich and Kononova 2003; Steyaert et al. 2003). Most research has focused on enzymes that degrade carbohydrates such as chitin, and the chitinolytic system of Trichoderma has been studied in detail (Kubicek et al. 2001). Chitinases are a family of enzymes, including exochitinases and endochitinases, that degrade chitin (Sharon et al. 2010). Nutrient starvation of the fungus and the products of chitin breakdown induce the chitinolytic enzyme expression system, whereas glucose and other simple sugars tend to suppress chitinolytic expression (Viterbo et al. 2002a). Although proteolytic enzymes have been less studied, a serine protease (Prb-1) produced by T. atroviride strain IMI 206040 inserted as multiple copies exhibited biological control potential against R. solani and also exhibited improved control potential against root-knot nematodes (Sharon et al. 2007). It is perhaps not surprising that fungal egg parasites that need to disrupt the eggshell probably require a combination of both chitinolytic and proteolytic enzymes (Morton et al. 2004).

4.2.3 Antibiotics

Trichoderma species can produce a variety of antibiotic compounds, which may contribute to the biological control processes. The nature and roles of antibiotic peptides that belong to the peptaibol group have been intensively studied (Szekeres et al. 2005). Peptaibols generally exhibit antimicrobial activity, which is thought to arise from their ability to form pores in lipid membranes. A peptaibol synthetase gene has been cloned (Wiest et al. 2002) and further studies suggested that peptaibols are critical in the chemical communication between Trichoderma and plants as triggers of non-cultivar-specific defence responses (Viterbo et al. 2007). Trichoderma virens produces gliotoxin and gliovirin and also peptaibols (Howell 2003). The antifungal action of enzymes reinforced by synergism with antibiotics was comprehensively reviewed by Kubicek et al. (2001) and this again points to multiple modes of action combining to generate functionality. Nematicidal activity against M. javanica J2s was detected in T. atroviride culture filtrates (CFs) and the active component/s had low molecular weight (MW) and heat sensitivity. Immature eggs, exposed to CFs exhibited reduced hatching rates, whereas hatching of mature eggs was enhanced. The effect of CF on eggs was caused by both the enzymatic fraction, which contained proteases and chitinases, and by the low molecular mass component/s. (Sharon, Chet and Spiegel unpublished). Appropriate candidates responsible for such nematicidal activity might be antibiotic peptides, such as peptaibols (Sharon et al. 2007).

4.2.4 Fungal Genomics

Utilizing genomic and metabolomic means to understand Trichoderma-plant disease interactions (Shoresh and Harman 2008; Woo et al. 2009) can further enhance the efficacy of Trichoderma as a biocontrol agent against nematode pests. Comparative genomics and maximum likelihood based methods showed that three different chitinase subgroups have expanded in copy number in Trichoderma species, indicating an important role of these chitinases during the mycoparasitism process (Ihrmark et al. 2010). Several regions and amino acids have been identified in four chitinase genes from different Trichoderma species, that are likely to determine functional properties playing an important role in the interaction between plant/pathogens and mycoparasites, such as substrate-specificity, processivity or pH-optima. These approaches can help lead to a better understanding of multitrophic nematode/plant/Trichoderma interactions and improvement of the biocontrol activity.

4.3 Pasteuria penetrans Genomics Applied to Culturing and Host Specificity

Pasteuria penetrans is an endospore-forming Gram positive bacterium that is part of a group of invertebrate parasites that infect nematodes and water fleas (Daphnia spp). One of the members of this group, P. ramosa, which is a parasite of Daphnia, has become a model for studying host-parasite co-evolution (Decaestecker et al. 2007) and innate immunity (Little et al. 2003; Kurtz 2005). Within the plant-parasitic nematode context, research over the last twenty years has focused on P. penetrans and its potential to be developed into a biological control agent of plant-parasitic nematode pests (Davies 2009). The Pasteuria group is phylogenetically closely related to Bacillus and Clostridium spp. (Charles et al. 2005) and there are a number of different species, the designations of which are determined by descriptions of morphology, host-range, life-cycle and, more recently, 16s rDNA sequence (Table 23.1). The two main features of this bacterium that have prohibited its commercial development are its host specificity and the inability to mass produce it in vitro.

Table 23.1 Species of Pasteuria so far described and their invertebrate host
Full size table

4.3.1 Genomic insights for In Vitro Culturing

Mass production of P. penetrans has had to rely on in vivo culturing methods of which the majority are adaptations of the method developed by Stirling and Wachtel (1980). Briefly, infective root-knot nematode juveniles are exposed to endospores so that each juvenile is encumbered with 5–10 endospores. These encumbered second-stage juveniles are then placed around the roots of a tomato plant at 25°C. After 6–8 weeks the tomato roots containing nematodes infected with spores are washed free of soil and air dried. The roots are then milled and can be used as inoculum for application to soil. Such milled tomato root can contain as many as 1.3 ´ 109 endospores per gram of root powder (Pembroke and Gowen, personal communication). Early attempts to grow Pasteuria in vitro produced very limited success. However, more recently, Pasteuria Bioscience LLC, Florida, has developed a system for in vitro mass culture of Pasteuria (Hewlett et al. 2004). Their system can mass produce different species of Pasteuria and Pasteuria usage has been cleared by the Environmental Protection Agency (2009) for the control of sting nematode (Belonolaimus longicaudatus) on golf courses in the United States.

One of the problems encountered in culturing Pasteuria in vitro has been getting vegetative colonies to sporulate and comparative genomics has helped to shed light onto this problem. Sporulation in Bacillus subtilis has been extensively studied and is dependent on a phosphorelay pathway (Burbulys et al. 1991) in which a phosphoryl group is transferred to the regulator Spo0F through a group of five kinases that are under environmental regulation. Like all known regulators of this type, Spo0F requires a divalent metal ion to be present in the conserved aspartic acid pocket in order for phosphorylation to occur (Grimshaw et al. 1998) and Mg2+ has been shown to be important for this process (Zapf et al. 1996). More recently it has been suggested that metal cations may play a role in the structure and function of Spo0F and its involvement in the initiation of sporulation (Mukhopadhyay et al. 2004). Investigations of the effects of the divalent cations Ca2+, Cu2+, Mg2+, and Mn2+ on the structure and function of B. subtilis Spo0F showed that they bound to the aspartic acid pocket and that while Mg2+ supports phosphotransfer from the kinase KinA to Spo0F the copper cation Cu2+ inhibited their phosphotransfer (Kojetin et al. 2005).

Interrogation of the Pasteuria survey sequence revealed genes with a high degree of similarity to genes involved in sporulation (Bird et al. 2003) and this included Spo0F. Alignment of Spo0F between B. subtilis, B. anthracis and B. thuringiensis and P. penetrans showed that key amino acids are conserved across these species. From the results discussed above it was hypothesised that the presence of Cu2+, at non-lethal concentrations in the sporulation media for B. subtilis and the related bacterium P. penetrans, might inhibit endospore formation while continuing to permit vegetative growth. Indeed, subsequent experiments revealed that the absence of Cu2+ in the media resulted in an increased number of sporulating cells (Kojetin et al. 2005). This result suggests that the availability of Cu2+ could be used to induce vegetative cells to enter sporulation.

4.3.2 Host Specificity and Endospore Attachment

The first stage of infection of a nematode by Pasteuria is when an endospore adheres to the surface of the nematode cuticle, and as discussed above, the Red Queen Hypothesis (Van Valen 1973, 1976) would suggest that within the context of an arms race it might be expected that functional diversity would co-evolve. Indeed, one isolate of P. penetrans does not adhere equally well to all populations of root-knot nematodes and the process of initial attachment is not linked to the phylogenetic diversity of the nematode populations tested (Davies et al. 2001). Five different monoclonal antibodies (Mabs) were raised to Pasteuria strain PP1 endospores from a single host female. Studies using these Mabs showed that there was a diversity of surface types as different sub-populations of the endospores of strain PP1 were recognised by each of the five different Mabs (Davies et al. 1994). Baiting the PP1 population of endospores with different species and races of root-knot nematode and using the Mabs to characterise the endospores that were adhering to each of the different populations of nematode, showed that different sub-populations of the endospores were adhering to the different nematode populations. This indicates that immunological heterogeneity in the surface of the endospore was related to heterogeneity present in the outer surface coat of the different nematode populations (Davies et al. 1994). Similar differences were also observed in the recognition of the surface of endospores between isolates of Pasteuria from different geographical regions by the different Mabs. One particular Mab, PP1/117, appeared to recognise the concave surface of the endospore to a greater extent than the upper surface, revealing that the density of the antigen was greater on the concave surface; pre-treatment of these endospores with sugars or glycolytic enzymes reduced the ability of the Mab to bind, suggesting the Mab was recognising a carbohydrate epitope (Davies and Redden 1997).

The biochemical mechanism by which endospores of Pasteuria adhere to the nematode cuticle is poorly understood; however, it has been proposed that carbohydrate-lectin type interactions may be involved (Davies and Danks 1993). In a genomic survey sequence of Pasteuria, collagen-like genes were identified (Davies and Opperman 2006) which were initially thought to be contaminating genes from the nematode itself. However, subsequent analysis showed them to be similar to collagen-like genes identified in other closely related Bacillus species (Sylvestre et al. 2002, 2003, 2005) and similar genes, that appear to be highly polymorphic, have also been found in P. ramosa (Mouton et al. 2009). It has been suggested that these genes form fibres on the surface of the endospore and are important in attachment to the nematode cuticle (Davies 2009). If collagen-like fibres on the surface of the endospore are involved in attachment, what are the molecules involved on the surface of the nematode? Mucins are a family of polypeptides associated with both the innate and adapted immune systems (Strous and Dekker 1992), which possess a polypeptide backbone that is highly glycosylated with sugar side chains. Glycosylation is predominantly O-linked through N-acetylgalactosamine (GalNAc) to serine and threonine residues within a variable number of tandem repeats (VNTR) regions of the polypeptide core (Hicks et al. 2000; Theodoropoulos et al. 2001). Although there is no information on the role of mucins in plant-parasitic nematodes, it is interesting that the genes muc-2, muc-3 and muc-4, that are members of the TES-120 family of proteins present in Toxocara canis (Tetteh et al. 1999) and are responsible for surface coat variation and have homologues in C. elegans, are also all present in various species of plant-parasitic nematodes (Davies 2009).

The investigations outlined above have been brought together in a hypothesis which suggests that collagen-like proteins on the surface of the endospore are interacting with mucin-like molecules present on the surface coat of the infective juvenile in what has been described as a ‘Velcro’-like mechanism (Davies 2009). Although the details of this system are yet to be elucidated, it is clear that both collagen-like molecules in respect to the endospore and mucin-like molecules with respect to the surface coat of the nematode would perfectly fit within the context of Van Valen’s (1976) Red Queen Hypothesis.

5 Future Developments

Over the last two decades since the publication of Stirling’s (1991) book on biological control, biology has undergone a revolution that combines the technologies of inexpensive and rapid DNA sequencing together with ‘in silico’ computing developments in the growing field of genomics. The purpose of this chapter has been to try and clarify some of the ecological constraints that have prohibited the development of biological control from being a robust strategy to control nematode pests, by exploring some of the molecular and biochemical mechanisms that underpin host-parasite interactions. These areas can be brought together in the new and rapidly developing field of ecological genomics, which is multidisciplinary and integrates a number of disciplines (Fig. 23.1). By taking such an approach genomics can act as a catalyst for enabling ecology to become an increasingly hypothesis-driven area of research, and bring unity to these formally disparate areas of study. Whereas formerly gene function was analysed through knocking out genes, and was a fairly blunt tool, comparative genomics can look at natural genetic variation across ecoclines and examine how this plays a role in the functional ecology of host-parasite interactions. Therefore, from following a simple reductionist approach that runs from the gene, up through proteins and biochemical pathways to cells, tissues and organisms, a more integrated, holistic approach can be undertaken in which downward causation from communities of populations selected by the environment, lead to the structuring of particular genotypes engaged in multitrophic interactions between plant-hosts, nematode-pests and their parasites (Davies and Spiegel 2010).

Fig. 23.1
figure 1

Disciplines that need to be integrated within the context of ecological genomics as a strategy for the development of an understanding of biological control of nematodes. (Adapted from Straalen and Roelofs 2006)

Full size image

5.1 Synthetic Biology

Very recently, the first microorganism was grown in which all the metabolic processes were controlled from an artificially constructed genome (Gibson et al. 2010). This arguably heralds the beginning of the epoch of synthetic biology, but the vision of designer organisms was first articulated in the 1970s when it was stated (Szybalski 1974):

But the real challenge will start when we enter the synthetic biology phase of research in our field. We will then devise new control elements and add these new modules to the existing genomes or build up wholly new genomes.

This naturally begs the question as to whether the development of designer biological control agents is a future possibility. To date, the isolation, mass production and re-introduction of potential biological control agents has only met with limited success, but the idea of construction de novo of microorganisms with exactly the functionality required offers a multitude of possibilities. Although such approaches are in the medium to long term future, the development of this type of technology will not be beyond criticism, and many of the societal issues that confront the development of genetically modified crops will also need to be addressed regarding designer biological control agents (Davies and Spiegel 2010).

5.2 Commercial Development and Future Directions

In the relatively short term biological control will continue to offer products that will be of value to niche markets (Whipps and Davies 2000; Gowen et al. 2007; Hallman et al. 2009). However, the commercial market for the control of plant-parasitic nematodes in arable crops is immense and with the continued withdrawal of nematicides from the market due to legislation, the challenge to produce alternatives will remain. The continued study of biological control is important, not only for developing robust agents that can be applied and be expected to work with confidence, but also in the long term, in the epoch of synthetic biology, new combinations of functional traits can be assembled in novel systems that so far are unimaginable.