Attachment tests of Pasteuria penetrans to the cuticle of plant and animal parasitic nematodes, free living nematodes and srf mutants of Caenorhabditis elegans

Populations of Pasteuria penetrans isolated from root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp.) were tested for their ability to adhere to a limited selection of sheathed and exsheathed animal parasitic nematodes, free living nematodes, including Caenorhabditis elegans wild type and several srf mutants, and plant parasitic nematodes. The attachment of spores of Pasteuria was restricted and no spores were observed adhering to any of the animal parasitic nematodes either with or without their sheath or to any of the free living nematodes including C. elegans and the srf mutants. All spore attachment was restricted to plant parasitic nematodes; however, spores isolated from cyst nematodes showed the ability to adhere to other genera of plant parasitic nematodes which was not the case with spores isolated from root-knot nematodes. The results are discussed in relationship to cuticular heterogeneity.


Introduction
The control of human and animal nematode infections is largely based on the administration of anthelmintic drugs. However, in response to the intensive use of anthelmintics, resistance has been reported (Waller, 1990;Jackson, 1993;De Clercq et al., 1997;Reynoldson et al., 1998) and this has led to a search for alternative strategies.
Most research on P. penetrans has concentrated on its interaction with root-knot nematodes (Meloidogyne spp.) and it has been shown to have potential to control root-knot nematodes (Stirling, 1991). The life cycle of P. penetrans commences when endospores attach to the cuticle of motile larvae as they migrate through the soil. Spores either germinate and penetrate the secondstage juvenile before the nematode has infected a plant root, as in the case of spores adhering to the cuticle of Heterodera avenae (Davies et al., 1990), or after the nematode has infected a plant root and started feeding (Sayre & Starr, 1985, 1988. In both cases, the spores each produce a germ tube which penetrates the nematode cuticle and produces a dichotomously branched microcolony. These microcolonies subsequently divide and proliferate throughout the pseudocoelom eventually killing the nematode and producing a cadaver filled with spores (Sayre & Starr, 1988). Pasteuria spores have been shown to differ in their ability to adhere to the cuticle of plant parasitic nematodes (Davies et al., 1988;Stirling, 1991) and the interaction between the nematode and Pasteuria is thought to involve a protein/carbohydrate like mechanism between the spore and the nematode cuticle (Davies et al., 1994). There are no reports of isolates of the bacteria being tested against either animal parasitic or free living nematodes, and Caenorhabditis elegans represents the latter catagory where mutants are available which differ solely in the surface characteristics of their cuticle. This paper reports the results of tests to study the attachment of spores, of selected Pasteuria isolates, to different species of animal parasitic (with and without sheath) and free living nematodes, to plant parasitic nematodes and surface (srf) mutants of Caenorhabditis elegans, the cuticles of which react differently to either antibodies and/or lectins.

Nematodes
The plant-parasitic nematodes Meloidogyne spp. and Rotylenchulus reniformis were obtained from plant cultures maintained in the glasshouse at 25ЊC on tomato plants, cv. Pixie, grown in a peat/sand (1:1, v/v) compost. Pratylenchus spp. and Radopholus similis were obtained from axenic maize root cultures (Hooper, 1986b). Nematodes were hatched from infected root material by placing small samples of infected root material in tap water on a small sieve in a tray of water at room temperature (Hooper, 1986a). The juveniles of cyst forming nematodes were obtained by incubating cysts at optimum temperatures in tap water and, in the case of the two species of potato cyst nematode, Globodera pallida and G. rostochiensis, in the presence of potato root diffusate. Aphelenchoides sp. and Ditylenchus sp were obtained from axenic Petri dish cultures of Botrytis cinerea maintained at room temperature in the laboratory by washing the surface of the agar with water (Hooper, 1986b). Samples of Anguina tritici were obtained by breaking open infected grains of wheat in a small drop of tap water to release the nematodes (Hooper, 1986a).
Cultures of the animal parasitic nematodes Haemonchus contortus, Ostertagia (Teladorsagia) circumcincta and Trichostrongylus axei were provided by Drs E. Munn, Babraham Institute, Cambridge, and R. Coop, The Moredun Research Institute, Edinburgh. Ancylostoma ceylanicum and Heligmosomoides polygyrus were obtained from the faecal material of infected hamsters and mice respectively (Garside & Behnke, 1989). Steinernema and Heterorhabditis were cultured in Galleria larvae. The freeliving nematodes Panagrellus redivivus, Pelodera strongyloides, Diplogaster sp., Mesodiplogaster sp., Panagrolaimus sp., and Rhabditis sp. were obtained from axenic Petri dish cultures maintained at room temperature in the laboratory. The wild type culture of Caenorhabditis elegans (N2) was provided by Dr Julie Arhinger, Medical Research Council, Cambridge and the surface mutants AT6, AT10 and CL261 (table 2) were obtained from Dr Theresa Stiernagle, Caenorhabditis Genetics Center, University of Minnesota and were maintained in Petri dishes seeded with E. coli strain OP50 following the method of Wood (1988).

Bacterial cultures
Populations of Pasteuria were obtained from the species of nematode from which they were originally isolated growing on a suitable host plant. Either, the infected roots were dried and the powder produced following the method of Stirling & Wachtel (1980), or Pasteuria infected nematode cadavers were collected from field soils. The latter were recognized using a dissecting microscope and identifying females present on or in roots but not producing egg masses (Sharma & Davies, 1996). Suspensions of spores were prepared by grinding either Pasteuria infested root powder, or infected cadavers, in tap water with a pestle and mortar. The spores were filtered with a 10 mm sieve, counted using a haemocytometer slide and the concentrations of suspensions were adjusted to 10 6 spores/ml. Stock suspensions were stored frozen at ¹20ЊC.

Attachment tests
Samples (250 ml) of spore suspensions of each of the stock Pasteuria populations were placed in separate siliconized Eppendorf tubes together with a 250 ml of a suspension of the test nematode population containing approximately 500 individuals. The nematodes and spores were thoroughly mixed and an attachment test performed by centrifugation (10 000 g for 5 min) following the method of Hewlett & Dickson (1993). A semi-quantitative score (0, no spores per nematode; þ, 1-10 spores per nematode; þþ, 11-40 spores per nematode) was given for each population of nematode tested, assessing a minimum of 25 nematodes for each nematode population, using a light microscope (× 400).

Results and discussion
No Pasteuria spores were observed adhering to any of the 3rd stage infective larvae of the animal parasitic nematodes either with or without their sheath (table 1) or to any of the free living nematodes including C. elegans and three srf mutants (table 2). All populations of Pasteuria used in these experiments had been isolated from plant parasitic nematodes and their attachment was restricted to plant parasitic nematodes (table 3). Attachment of those populations of spores isolated from rootknot nematodes (Meloidogyne spp.) was similarly restricted to root-knot nematodes, however, those isolated from the genus Heterodera appeared to have a broader range of hosts and all three Pasteuria populations, PPC, PPN and PPW also attached to Globodera. One population of spores, PPC, was also observed attaching to Pratylenchus, Radopholus, Rotylenchulus and Aphelenchoides; the attachment of these spores also exhibited interspecific variation between species within genera (table 3). It is interesting to note that the populations of Pasteuria from the apomictic populations of nematodes, i.e. the root-knot populations, appear to have a more restricted host range than those isolated from the cyst nematode populations which are amphimictic.
There are two fundamental problems in the deployment of Pasteuria as a biological nematicide, firstly, the inability to culture large populations of spores (Williams et al., 1989;Bishop and Ellar, 1991) and secondly, its host specificity (Stirling, 1985;Channer & Gowen, 1992;Davies et al., 1988). Populations of Pasteuria are found which parasitize all the major genera of plant parasitic nematodes (Sayre and Starr, 1988) and there have recently been reports of other populations which parasitize nematodes in other families and even orders (Sturhan, 1996). Monoclonal antibodies have shown that the surface of the spores of a Pasteuria isolate originating from M. incognita race 2 was highly heterogeneous, and baiting experiments showed that different sub-populations of spores adhere to different species and races of nematode (Davies et al., 1994). These and subsequent studies (Davies & Redden, 1997) have suggested that the surface properties of the spore are responsible for the virulence of the bacterium and suggest that similar heterogeneity will also be present in the nematode cuticle. As the bacterium infects other invertebrates such as the cladoceran Moina   (Sayre et al., 1977;Ebert et al., 1996) it would seem likely that similar bacteria will be found infecting animal parasitic nematodes, especially those which have to spend prolonged periods of their life cycle in soil before infecting their respective animal hosts, and these infective stages will also exhibit a high level of cuticular heterogeneity. The challenge for the future will therefore be to isolate such bacteria targeting animal and human parasitic nematodes, and to evaluate their potential as tools for the biological control of important human and livestock diseases. Meloidogyne incognita IACR þþ þ þþ