Ultrastructure and Development of Pasteuria Sp. (S-1 Strain), an Obligate Endoparasite of Belonolaimus Longicaudatus (Nemata: Tylenchida) R
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Journal of Nematology 33(4):227–238. 2001. © The Society of Nematologists 2001. Ultrastructure and Development of Pasteuria sp. (S-1 strain), an Obligate Endoparasite of Belonolaimus longicaudatus (Nemata: Tylenchida) R. M. Giblin-Davis,1 D. S. Williams,2 W. P. Wergin,3 D. W. Dickson,4 T. E. Hewlett,4 S. Bekal,5 and J. O. Becker5 Abstract: Pasteuria sp., strain S-1, is a gram-positive, obligate endoparasitic bacterium that uses the phytoparasitic sting nematode, Belonolaimus longicaudatus, as its host in Florida. The host attachment of S-1 appears to be specific to the genus Belonolaimus with development occurring only in juveniles and adults of B. longicaudatus. This bacterium is characterized from other described species of Pasteuria using ultrastructure of the mature endospore. Penetration, development, and sporogenesis were elucidated with TEM, LTSEM, and SEM and are similar to other nematode-specific Pasteuria. Recent analysis of 16S rDNA sequence homology confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans, and corroborates ultrastructural, morphological, morphometric, and host-range evidence suggesting separate species status. Key words: Belonolaimus longicaudatus, development, obligate nematode endoparasitic bacterium, Pasteuria sp. (S-1 strain), sporo- genesis, sting nematode, ultrastructure. Investigators have identified four nominal species of al., 1989). Thus, traditional procedures for biochemical Pasteuria that are gram-positive, mycelial, endospore- characterization have not been available for elucidation forming, and endoparasitic on nematodes and crusta- of Pasteuria species (Sayre and Starr, 1985). The four ceans. The type species, Pasteuria ramosa, parasitizes wa- nominal species of Pasteuria were described using a Lin- ter fleas (Cladocera; Daphinidae) (Ebert et al., 1996; nean species concept that relied upon morphometrics, Metchnikoff, 1888; Sayre et al., 1979, 1983), while the ultrastructural comparisons of mature endospores, and other three species are reported in association with host attachment trials. Cianco (1995) challenged the phytoparasitic nematodes. Pasteuria penetrans parasitizes reliability of morphometrics and host range for Pasteu- root-knot nematodes, Meloidogyne spp. (Mankau, 1975; ria taxonomy. Numerous potential Pasteuria species, Sayre and Starr, 1985; Starr and Sayre, 1988); P. thornei which exist in soil and freshwater habitats, may overlap parasitizes root-lesion nematodes, Pratylenchus spp. considerably in morphometrics and host range (Chen (Starr and Sayre, 1988; Sayre et al., 1988); and P. nish- and Dickson, 1998). Ultrastructure of mature endo- izawae parasitizes the cyst nematodes, Heterodera and spores appears to provide reliable traits in the few spe- Globodera (Sayre et al., 1991b). Pasteuria strains are glo- cies of Pasteuria that have been named, especially when bally distributed and have been reported attached to used in combination with morphometrics and host and parasitizing numerous nematode species (>300) range (Ebert et al., 1996; Sayre et al., 1991a, 1991b; from different trophic groups including plant-parasitic, Sayre and Starr, 1985; Starr and Sayre, 1988). However, predatory, insect-parasitic, and free-living nematodes this still leaves a dearth of taxonomic characters with (Atibalentja et al., 2000; Cianco et al., 1994). The biol- phylogenetic value. Recent phylogenetic analyses of ogy and taxonomy of Pasteuria have been obscure and 16S rDNA sequences from P. ramosa (U34688; Ebert et difficult to study because of their small size and highly al., 1996), P. penetrans (AF077672; Anderson et al., host-specific and obligate endoparasitic nature (Chen 1999; Bekal et al., 2001), Pasteuria sp. ex Heterodera gly- and Dickson, 1998; Dickson et al., 1994; Ebert et al., cines (AF1 34868; Atibalentja et al., 2000), and Pasteuria 1996; Giblin-Davis, 2000; Giblin-Davis et al., 1990). sp., strain S-1 ex Belonolaimus longicaudatus (AF254387; Monoxenic culture attempts have been unsuccessful Bekal et al., 2001) have generated hypotheses about the due to a lack of knowledge about these fastidious bac- placement of the genus Pasteuria within the bacterial teria and the physiology of their nematode hosts kingdom, with relationship to the genus Alicyclobacillus (Bishop and Ellar, 1991; Riese et al., 1988; Williams et (Atibalentja et al., 2000; Ebert et al., 1996) and pro- vided independent evidence concerning species rela- tionships that can be used with ultrastructural and host Received for publication 05 February 2001. specificity data. 1 University of Florida-IFAS, Fort Lauderdale Research and Education Cen- We examined the development and ultrastructure of ter, 3205 College Ave., Davie, FL 33314-7799. 2 Microbiology and Cell Science, University of Florida, PO Box 110700, the mature endospore of a putative new species of Pas- Gainesville, FL 32611-0700. teuria (S-1 strain) from the sting nematode, B. longicau- 3 Nematology Laboratory, USDA, ARS, Bldg. 011-A, BARC-West, Beltsville, MD 20705. datus from Florida, previously referred to as an isolate 4 Department of Entomology and Nematology, University of Florida-IFAS, of the Pasteuria penetrans group, Pasteuria sp., or Pasteu- PO Box 110620, Gainesville, FL 32611-0620. 5 Department of Nematology, University of California, Riverside, CA 92521. ria n. sp. (S-1) (Bekal et al., 1999, 2001; Dickson et al., E-mail: giblin@ufl.edu 1994; Giblin-Davis, 1990, 2000; Giblin-Davis et al., 1990, The authors thank Henry Aldrich, Janete Brito, Barbara J. Center, Z. X. Chen, Billy Crow, and James F. Preston for help, discussions, and ideas usedin 1995). this project and Monica Elliott and Nigel Harrison for review of the manuscript. aterials and ethods This research was supported by a grant from the United States Golf Associa- M M tion. This manuscript is Florida Agricultural Experiment Stations Journal Series R-07848. Sources of S-1 Pasteuria: Belonolaimus longicaudatus were This paper was edited by Patricia Timper. collected from soil around the roots of bermudagrass 227 228 Journal of Nematology, Volume 33, No. 4, December 2001 (Cynodon spp.) grown in plots at the Fort Lauderdale Transmission electron microscopy of S-1 Pasteuria: S-1- Research and Education Center, Davie, Florida (26°05Ј infected specimens of B. longicaudatus for study of ma- 12ЉN 080°14Ј 26ЉW). The nematodes were separated ture endospore ultrastructure or changes during pen- from the soil using the sugar-flotation-centrifugation etration, development, and sporogenesis were cut into method (Jenkins, 1964). The S-1 strain had previously two or more pieces and fixed in 2% (v/v) glutaralde- been collected from this site (Bekal et al., 1999, 2001; hyde, 2% (v/v) formaldehyde, in 0.1 M sodium caco- Giblin-Davis, 2000; Giblin-Davis et al., 1990, 1995). Live dylate buffer (pH 7.2) overnight at 4 °C, embedded in nematodes were examined using an Olympus BH-2 2 to 3% (w/v) agarose, and cut into small blocks. The photomicroscope fitted with Nomarski optics and a glutaraldehyde was rinsed from the blocks with five camera lucida to identify the stage of nematode, attach- rinses of 0.1 M sodium cacodylate buffer, and the tissue ment of endospores to the cuticle, and presence of was postfixed in 2% (w/v) OsO4 in 0.1 M sodium cac- different stages of internal parasitism. Crystal violet odylate buffer and 1% (w/v) aqueous uranyl acetate. staining (<1%; w/v) was used to increase visibility of The tissue was dehydrated in an ethanol-acetone series, externally attached spores. Endospore measurements and embedded in Spurr’s epoxy resin. Thin-sections were made from samples of spores from five different were cut with a diamond knife on an LKB Ultratome III nematodes collected from this site. Counts of mature or an RMC MT-6000-XL ultramicrotome, stained with endospores from spore-filled cadavers were done by uranyl acetate and lead citrate, and viewed with a Hita- collecting different stages of B. longicaudatus that were chi H-7000 or a Zeiss-EM10 (Thornwood, NY) TEM. filled with spores, squashing them individually in a drop of water, then counting the spores in a set volume Results and Discussion on a hemocytometer. In addition, B. longicaudatus with apparent endospore infestations of S-1 from soil The ultrastructure of the mature endospore of S-1 is around bermudagrass roots on a golf course in Or- distinctive when compared with endospores of the lando, Florida, and from soil around potato plants in named species of Pasteuria (Giblin-Davis, 2000; Giblin- Hastings, Florida, were collected and processed for Davis et al., 1990, 1995). The terminology used herein transmission electron microscopic (TEM) comparisons to describe endospore morphology was previously de- with the Davie, Florida isolate. fined by Sturhan et al. (1994). With SEM and LTSEM, Conventional and low-temperature scanning electron mi- peripheral fibers of the mature endospore unevenly croscopy of S-1 Pasteuria: We examined the external mor- protrude around the exposed spherical outer coat of phology of S-1 from Davie, Florida, with conventional the spore creating a crenate border (Fig. 1B–D) that is scanning electron microscopy (SEM) and low- similar to that observed for a Pasteuria strain from Ho- temperature scanning electron microscopy (LTSEM). plolaimus galeatus from Peru (Cianco et al., 1998). The For SEM, specimens of B. longicaudatus with attached exosporium and sporangium can be retained upon spores were fixed in 3% (v/v) glutaraldehyde, dehy- spore attachment to the host cuticle (Fig. 1B) or be lost, drated in a graded ethanol series, critical point dried exposing the microfibrillar surface coat on the dorsum from liquid CO2, mounted on a stub with double sticky of the peripheral fibers (Fig. 1D), similar to P. penetrans tape, sputtercoated with 20 nm of gold-palladium, and (Chen et al., 1997; Sayre and Starr, 1985) and P. nish- viewed with a Hitachi (Tokyo, Japan) S-4000 Field Emis- izawae (Sayre et al., 1991a, 1991b). sion SEM at 7 kV. For LTSEM, live B. longicaudatus with TEM observations of mature endospores of S-1 inside attached spores were placed onto tissue paper and the host (Figs. 2, 3) and attached to the cuticle of a host mounted onto a stub.