‘Candidatus Pasteuria usgae’ sp. nov., an obligate endoparasite of the phytoparasitic nematode Belonolaimus longicaudatus

There are four nominal species of Pasteuria that are Gram-positive, mycelial, endospore-forming and endoparasitic on nematodes and crustaceans. Pasteuria ramosa (type species) has been described from water fleas (Cladocera: Daphnidae) (Ebert et al., 1996; Metchnikoff, 1888; Sayre et al., 1979, 1983). The other three species are associated with phytoparasitic nematodes. Pasteuria penetrans has been described from root-knot nematodes (Meloidogyne spp.) (Sayre & Starr, 1985; Starr & Sayre, 1988), Pasteuria thornei has been reported from root-lesion nematodes (Pratylenchus spp.) (Starr & Sayre, 1988; Sayre et al., 1988) and Pasteuria nishizawae was found parasitizing cyst nematodes of the genera Heterodera and Globodera (Sayre et al., 1991). Pasteuria strains have been reported attached to and parasitizing numerous nematode species (>300) from around the world (Atibalentja et al., 2000; Ciancio et al., 1994). They are difficult to study because of their highly host-specific and obligately endoparasitic nature (Chen & Dickson, 1998; Dickson et al., 1994; Ebert et al., 1996; Giblin-Davis, 2000; Giblin-Davis et al., 1990), which has prevented successful culture in vitro (Bishop & Ellar, 1991; Riese et al., 1988; Williams et al., 1989). Traditional procedures for biochemical characterization are not available for elucidation of Pasteuria species (Sayre & Starr, 1985). The four nominal species of Pasteuria were described using the Linnaean species concept based upon discontinuities in morphometrics, ultrastructure of mature endospores and host attachment specificity. The ultrastructure of the mature endospore of strain S-1, a Gram-positive, obligately endoparasitic prokaryote that uses the phytoparasitic sting nematode Belonolaimus longicaudatus as its host, is distinctive when compared with endospores of named species of Pasteuria (Giblin-Davis, 2000; Giblin-Davis et al., 1990, 1995, 2001). The terminology used herein to describe endospore morphology was previously defined by Sturhan et al. (1994).

P. ramosa Metchnikoff 1888 was described from the water fleas Daphnia magna and Daphnia pulex. All attempts at culture failed and a type strain was not established (Metchnikoff, 1888). Years of confusion ensued, as the original classification of P. ramosa as a bacterium was challenged with suggestions that it was a Torula yeast, a microsporidian or a member of the haplosporidian genera Democystidium or Lymphocystidium (Ebert et al., 1996). The situation was complicated further when Hirsch (1972) and Staley (1973) used the name P. ramosa Metchnikoff 1888 for a superficially similar budding bacterial species found on the exterior surfaces of Daphnia species. This prokaryote (strain ATCC 27377) was cultivated in vitro and erroneously assigned as the type species of the genus Pasteuria, even though it did not form endospores, mycelia or branches, was not an endoparasite of cladocerans and had a Gram-negative reaction (Staley, 1973). Strain ATCC 27377 was subsequently reclassified as Planctomyces staleyi Starr, Sayre & Schmidt 1983 (Starr et al., 1983). The latter authors requested conservation of the original description of P. ramosa Metchnikoff 1888 and rejection of ATCC 27377 as the type strain. That request was supported by the Judicial Commission of the International Committee for Systematic Bacteriology (1986). Strain ATCC 27377 was then assigned to Pirella staleyi by Schlesner & Hirsch (1984), which did not have priority because a fungal genus already occupied the name Pirella. Thus, a new genus, Pirellula, was created with strain ATCC 27377 being named Pirellula staleyi (Schlesner & Hirsch 1984) by Schlesner & Hirsch (1987).

A P. ramosa-like strain was discovered infecting Moina rectirostris, a member of the Daphnidae (Sayre et al., 1977), and this strain was used in the emendation of the species (Starr et al., 1983). However, Ebert et al. (1996) have proposed that the Daphnia-parasitic P. ramosa they characterized from the same host as Metchnikoff (1888) be designated as the neotype for P. ramosa Metchnikoff 1888 and that the Moina isolate be compared directly to the neotype in future studies.⇓

Because the obligately endoparasitic nature of Pasteuria currently prevents isolation of a definitive type strain, ‘Candidatus’ status is proposed for each novel provisional species designation in this genus (see Murray & Schleifer, 1994; Murray & Stackebrandt, 1995; Stackebrandt et al., 2002). All of the currently named species in the genus Pasteuria Metchnikoff 1888 have nomenclatural standing and remain validly named species. These species are P. ramosa Metchnikoff 1888 (Approved Lists 1980) emend. Starr et al. 1986 [with the description of Metchnikoff (1888) as emended by Starr et al. (1983) serving as the type: see Judicial Commission of the International Committee on Systematic Bacteriology (1986); Wayne (1986)], P. nishizawae Sayre et al. 1992 [description and illustrations from Sayre et al. (1991) serving as type], P. penetrans (ex Thorne 1940) Sayre & Starr 1986 [description and illustrations from Sayre & Starr (1985) serving as type] and P. thornei Starr & Sayre 1988 [description and illustrations from Sayre et al. (1988) serving as type]. We concur with the proposal by Ebert et al. (1996) to accept the Daphnia parasite that they isolated, studied and sequenced as the neotype for P. ramosa and the genus Pasteuria. Unfortunately, 16S rDNA sequence data are not available for P. thornei or P. nishizawae and these forms must be rediscovered before a more complete characterization of these Pasteuria and ‘Candidatus Pasteuria’ species can be made.

Recent phylogenetic analyses of 16S rDNA sequences from P. ramosa (U34688) (Ebert et al., 1996), P. penetrans (AF077672) (Anderson et al., 1999; Bekal et al., 2001), ‘Candidatus Pasteuria sp.’ strain HG ex Heterodera glycines (AF134868) (Atibalentja et al., 2000) and ‘Candidatus Pasteuria sp.’ strain S-1 ex B. longicaudatus (AF254387) (Bekal et al., 2001) have shown that Pasteuria is embedded in the same clade as members of the genus Alicyclobacillus (Atibalentja et al., 2000; Ebert et al., 1996). However, there is some support for Pasteuria as a separate clade originating between the Alicyclobacillus and Thermoactinomyces clades (Atibalentja et al., 2000). In addition, Pasteuria shared greatest sequence identity with Thermoactinomyces (about 87 %) (Atibalentja et al., 2000). 16S rDNA sequence analysis corroborates ultrastructural and host attachment studies that support a novel species designation for a ‘Candidatus Pasteuria’ collected from the sting nematode B. longicaudatus (Bekal et al., 2001; Giblin-Davis, 2000; Giblin-Davis et al., 2001). The 16S rDNA sequence corresponding to nucleotide positions 28–1390 of strain S-1 had similarity to previously published sequences of 96 % to P. penetrans (Anderson et al., 1999), 93 % to P. ramosa (Ebert et al., 1996) and 96 % to ‘Candidatus Pasteuria sp.’ strain HG of H. glycines (Atibalentja et al., 2000). Bootstrap analysis using maximum-likelihood, maximum-parsimony and minimum evolution showed strong support for a phytoparasitic nematode-associated clade (100 %) that excluded P. ramosa (Bekal et al., 2001). The two geographical strains of P. penetrans (Senegal and Florida) formed a robust clade (88–100 %), whereas strain S-1 was part of a weakly supported clade together with ‘Candidatus Pasteuria sp.’ strain HG of H. glycines (41–64 %) (Bekal et al., 2001). These data support the hypothesis that S-1 represents a novel species of ‘Candidatus Pasteuria’ (Bekal et al., 2001; Giblin-Davis, 2000; Giblin-Davis et al., 1990, 1995, 2001).

The name ‘Candidatus Pasteuria usgae’ sp. nov. is proposed for the ‘Candidatus Pasteuria sp.’ from the sting nematode B. longicaudatus from southern Florida [Fort Lauderdale Research and Education Center, Davie, Florida (26°05′12″N, 080°14′26″W)], previously referred to as an isolate of the P. penetrans group (Giblin-Davis, 1991; Giblin-Davis et al., 1990), a large-spored isolate of Pasteuria sp. from B. longicaudatus (Dickson et al., 1994), or as various designations involving Pasteuria and S-1 (Bekal et al., 1999, 2001; Brito et al., 2000; Giblin-Davis, 2000; Giblin-Davis et al., 1995, 1998, 2001). The description is based upon data and comparisons of S-1 with other described species of Pasteuria in three recently published papers (Bekal et al., 2001; Giblin-Davis, 2000; Giblin-Davis et al., 2001).