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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 83 (2003) 83–85 www.elsevier.com/locate/yjipa
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Morphology and ultrastructure of a Pasteuria form parasitic in Tylenchorhynchus cylindricus (Nematoda) In northeastern Spain, Tylenchorhynchus spp. is the second most abundant and frequent plant-parasitic nematode occurring in vegetable crops after Meloidogyne spp. (Ornat and Verdejo-Lucas, 1999; Sorribas and Verdejo-Lucas, 1994). A population of T. cylindricus naturally parasitized by a Pasteuria form was found in a tomato field at Cabrils, Barcelona (Spain). We report on the morphology and ultrastructure of this Pasteuria form investigated using light (LM), scanning (SEM), and transmission (TEM) electron microscopy. Composite soils samples were taken at week-intervals in June and July 2001 by collecting rhizosphere soil from tomato plants in each of four replicated plots (3:5 4 m), and nematodes were extracted immediately using Baermann trays. Temporary mounts in water were prepared for LM observation of endospores attached to and/or within the body cavity of T. cylindricus. A random sample of 30 individuals per replication and sampling time were examined at 200. Endospores from infected nematodes were released and their size, and the diameter of the central core measured with an eyepiece micrometer of a light microscope using a 100 oil immersion objective. For SEM studies, specimens with endospores attached to the cuticle were fixed overnight in 3% glutaraldehyde in cacodylate buffer 0.1 M at pH 7.2, dehydrated in an ethanol series, transferred to cover slips coated with 0.2% poly-L -lysine to allow nematode adhesion, and critical point dried in a Polaron CPD 7501. After transfer to SEM stubs, the specimens were sputter-coated with gold in a Biorad SC510. Endospores were observed with a Zeiss DSM 940A Scanning electron microscope at acceleration voltages of 10 kV. For TEM studies, specimens filled with endospores were fixed in 3% glutaraldehyde. Fixed material was washed several times with cacodylate buffer 0.1 M at pH 7.2, postfixed in 1% osmium tetraoxide and potassium ferrocyanide 0.8%, washed again, and embedded in 3% purified water agar. Blocks containing single nematodes were cut and dehydrated through an acetone series, before embedding in SpurrÕs medium (Spurr, 1969). Sections 80–90 nm thick were cut with a Leica ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Hitachi 800 MT Transmission electron microscope at 75 kV.
LM examinations of T. cylindricus showed Pasteuria sp. endospores attached to and/or filling the body (Fig. 1A) of larval stages, females, and males. The mean percentage of nematodes with endospores attached across all sampling periods was 8.6 and 2.6% of the individuals were filled with endospores. The endospore diameter was 4.2 lm and the central core measured 2.2 lm (Fig. 1B). Parasitized nematodes contained 340 35 endospores. SEM examinations showed the typical cup shape of attached endospores when visualized in dorsal view (Fig. 1C). TEM micrographs showed Pasteuria cells at different stages of sporogenesis filling the host body in a single section (Fig. 1D), the position of the germination aperture as a central reduction in the outer endospore coat (arrows) (Fig. 1E), and structural constituents of the endospore according to Imbriani and Mankau (1977) (Figs. 1F and G). The endospore showed symmetrical squared protuberances in the upper region (arrows heads) (Figs. 1D and E). The Pasteuria form from T. cylindricus appeared similar to other forms as concerns morphometric values with a medium size endospore diameter range from 3.5 to 5.0 lm (Ciancio, 1995; Ciancio et al., 1994), the mode of infection, and the general endospore organization. However, some peculiar characters were revealed by TEM as the endospore showed a particular shape characterized by angular protrusions of the distal core cover layers. The protuberances conferred to the endospore a characteristic angular shape, as seen in transverse sections (arrow heads) (Fig. 1F). The stranded material that remained in the matrix is another feature of this Pasteuria form together with the thickness of the parasporal fibres responsible for adhesion to the host. This is the first report of parasitism of T. cylindricus by a Pasteuria sp. from, although other species of the genus have been shown to be host of the bacterium including T. annulatus and T. maximus in US (Giblin-Davis et al., 1990; Sayre et al., 1988), T. dubius in Germany (Winkelheide and Sturhan, 1996), and T. leviterminalis in Japan (Talavera et al., 2001). The peculiar features of this Pasteuria form in T. cylindricus has not been reported, to our knowledge, in other species of this nematode or in other species of Pasteuria (Chen et al., 1997; Sayre and Starr, 1988; Starr and Sayre, 1988),
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Fig. 1. Light (A–B), SEM (C), and TEM (D–G) micrographs of Pasteuria sp. form parasitic in Tylenchorhynchus cylindricus. (A) Contemporary presence of attached endospores on the cuticle and within the body and penetration of cuticle by germ tube. (B) Mature endospores released from infected nematode. (C) Top view of the cup-shaped endospore. (D) and (E) Cross section through mid-body region of parasitized nematode showing different stages of sporogenesis, mature sporangia with fully developed endospores, the angular-squared organization of the upper region of the core walls (arrow heads), and the reduction of the outer endospore coat allowing the outgrowth of the nuclear germ peg during endospore germination (arrows). (F) Cross sections of endospore showing the nuclear core region (n), the cortex (c), a 160 nm thick outer cortical layer (occ), a 200 nm thick outer endospore coat (oec), and inner parasporal fibres (pf). (G) Section showing a coarse fibrillar matrix containing strand-like inclusions (ma), sporangial wall (sw), parasporal fibres (pf), basal parasporal fibres layer (bpf), and bilayered exosporium (ex). Scale bars: A, 4 lm; B, 12 lm; C–E, 1 lm; and F and G, 0.2 lm.
which suggests that this Pasteuria form may represent a distinct branch in the parasite radiation. The strict dependence of Pasteuria spp. on their corresponding
hosts may represent a suitable host-parasite model system to study factors influencing coevolution and divergence of species (Carius et al., 2001).
M. Galeano et al. / Journal of Invertebrate Pathology 83 (2003) 83–85
Acknowledgments Authors thank P. Castillo for nematode identification and M. Cermola, IIGBT-CNR, Naples, for assistance and suggestions. First author was supported by a grant from the Instituto Nacional de Investigaciones Agrarias (INIA), and Project No. SC98-103-C2-2. Second author acknowledges partial funding by MIPAF, PF ‘‘Orticoltura,’’ Publication No. 36.
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Magda Galeano Soledad Verdejo-Lucas Departament Protecci o Vegetal Institut de Recerca i Tecnologia Agroalimentaries Crta. de Cabrils s/n. 08348 Cabrils, Barcelona Spain E-mail address:
[email protected] Aurelio Ciancio Istituto per la Protezione delle Piante, Sezione di Bari Consiglio Nazionale delle Ricerche Via Amendola 165/A, 70126 Bari Italy
