Family V. Laurent 1890, 756AL Pas.teu.ri.a’ce.ae. N.L. fem. n. type genus of the family; suff. -aceae ending denoting family; N.L. fem. pl. n. Pasteuriaceae the family of Pasteuria. Gram-positive, dichotomously branching, septate mycelium, soil invertebrates. Has not been cultivated axenically, but can the terminal hyphae of which enlarge to form sporangia and be grown in the laboratory with its invertebrate host. eventually endospores. Nonmotile. Sporangia and microcolo- Type genus: Pasteuria Metchnikoff 1888, 166AL emend. Sayre nies are endoparasitic in the bodies of freshwater, plant, and and Starr 1985, 149; Starr and Sayre 1988a, 27.

Genus I. Pasteuria Metchnikoff 1888, 166AL emend. Sayre and Starr 1985, 149; emend. Starr and Sayre 1988a, 27 [Nom. Cons. Opin. 61 Jud. Comm. 1986, 119. Not Pasteuria in the sense of Henrici and Johnson (1935), Hirsch (1972), or Staley (1973); see Starr et al. (1983) and Judicial Commission (1986)]

RICHARD M. SAYRE AND MORTIMER P. STARR revised by DONALD W. DICKSON, JAMES F. PRESTON, III, ROBIN M. GIBLIN-DAVIS, GREGORY R. NOEL, DIETER EBERT AND GEORGE W. BIRD Pas.teu’ri.a. M.L. gen. n. Pasteuria of Pasteur, named after Louis Pasteur, French savant and scientist. Gram-positive, endospore-forming . Propagation fol- by Metchnikoff, this bacterium divides by budding. It forms a lowing germination within a or cladoceran host pro- major non-prosthecate appendage (a fascicle), it does not form ceeds through the formation of rounded to elliptical (termed endospores, it is not mycelial or branching, its staining reaction “cauliflower-like” by Metchnikoff, 1888) vegetative microcolo- is Gram-negative, and it is not an endoparasite of cladocerans. nies from which “daughter” microcolonies may be formed. The After searching for, but not finding, the bacterial endopara- sporogenous cells at the periphery of the colonies are usually site in water fleas as described by Metchnikoff, the erroneous attached by narrow “sacrificial” intercalary hyphae that lyse, conclusion was reached that this budding bacterium that occurs resulting in developing sporangia arranged in clumps of eight on the surfaces of was the organism Metch- or more, but more often in quartets, triplets, or doublets and, nikoff had described in 1888. As a result, a budding bacterium finally, as single teardrop-shaped or cup-shaped or rhomboi- (strain ATCC 27377) was mistakenly designated (Staley, 1973) dal mature sporangia. The rounded end of the sporangium as the type culture for Metchnikoff (1888), the encloses a single refractile endospore (1.0–3.0 µm in major type (and, then, sole) species of the genus Pasteuria. This confu- dimension), an oblate spheroid, ellipsoidal or almost spherical sion between Metchnikoff’s described cladoceran parasite and in shape, usually resistant to desiccation and elevated tempera- the quite different budding bacterium was resolved by Starr et al. tures (one species has somewhat limited heat tolerance). Non- (1983). The budding bacterium (with strain ATCC 27377 as motile. Sporangia and microcolonies are endoparasitic in the its type culture) was named Planctomyces staleyi Starr, Sayre and bodies of freshwater, plant, and soil invertebrates. Axenic cul- Schmidt (1983), now named Pirellula staleyi (Butler et al., 2002). tivation has not been documented, but it can be grown in the Conservation of the original descriptions of the genus Pasteu- laboratory with its invertebrate host. The pathogen is horizon- ria and Pasteuria ramosa, as updated, was recommended (Starr tally transmitted via soil or waterborne spores. Infected hosts et al., 1983) and approved (Judicial-Commission, 1986). Unfor- fail to reproduce. tunately, vestiges of this nomenclatural disorder remained for DNA G+C content (mol%): not known. some time. For example, certain evolutionary and taxonomic Type species: Pasteuria ramosa Metchnikoff 1888, 166AL. inferences (Woese, 1987) regarding the genus Pasteuria were based upon data concerning bacteria belonging to the Blasto- Further descriptive information caulis–Planctomyces group of budding and non-prosthecately A complex of errors initially confused our understanding of the appendaged bacteria rather than the mycelial and endospore- genus Pasteuria Metchnikoff (1888). Stated briefly, Metchnikoff forming invertebrate parasites that actually comprise the genus (1888) described an endospore-forming bacterial parasite of Pasteuria. A Pasteuria ramosa-like strain was discovered infecting cladocerans, which he named Pasteuria ramosa. Metchnikoff the cladoceran rectirostris, a member of the Daphniidae presented drawings and photomicrographs of the life stages of (Sayre et al., 1979), and this strain was used in the emendation this parasite as they occurred in the hemolymph of the water of the species (Starr et al., 1983). Ebert et al. (1996), however, fleas Leydig and Straus. He was, proposed that the Daphnia-parasitic Pasteuria ramosa that they however, unable to cultivate the organism in vitro. Subsequent had characterized from the same host as Metchnikoff (1888) be workers (Henrici and Johnson, 1935; Hirsch, 1972; Staley, designated the neotype for Pasteuria ramosa Metchnikoff (1888) 1973), who were looking in cladocerans for Metchnikoff’s and that the Moina isolate be compared directly to the neotype unique bacterium, reported on a different bacterium with in future studies. only superficial resemblance to certain life stages of Pasteuria Confusion of a different kind occurred in the nomenclature ramosa. Their investigations led to the axenic cultivation of a of the bacterial endoparasite of when Cobb (1906) budding bacterium that is occasionally found on the exterior erroneously reported the numerous highly refractive bodies surfaces of Daphnia species. Unlike the organism described infecting specimens of the nematode Dorylaimus bulbiferous

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as “perhaps monads” of a parasitic sporozoan. The incorrect However, penetrans was not included in the Approved placement in the protozoa of an organism now known to be Lists of Bacterial Names (Skerman et al., 1980), therefore it a bacterial parasite of nematodes has persisted for nearly 70 had no nomenclatural standing. Although this micro-organism years. Another incorrect placement was suggested by Mico- forms endospores of the sort typical of the genus Bacillus Cohn letzky (1925), who found a nematode parasite similar in size (1872), its other traits (e.g., mycelial habit, endoparasitic asso- and shape to those reported by Cobb. Micoletzky suggested that ciations with plant-parasitic nematodes) suggested that it did this organism belonged to the genus Duboscqia Perez (1908), not belong in the genus Bacillus. Rather, it is closely related to another sporozoan group (Perez, 1908). Later, Thorne (1940) Pasteuria ramosa (see Table 55 and Figure 37) and it has more presented a detailed description of a new parasite from the properly been assigned (Sayre and Starr, 1985) to the genus nematode pratensis (syn. Pratylenchus brachyurus, Pasteuria Metchnikoff (1888) as Pasteuria penetrans. see Sayre et al., 1988). Thorne assumed that this organism was The developmental stages that occur before sporulation are similar to the nematode parasite described by Micoletzky, similar for all Pasteuria spp. Except for Thermoactinomyces spp., thereby assigning it to the microsporidian genus Duboscqia, as the mycelial-like proliferation during development is not found Duboscqia penetrans. in any other endospore-forming bacteria. The morphological Thorne’s description and nomenclature persisted until 1975 changes that occur during sporulation appear to be generally even though other investigators (Canning, 1973; Williams, similar for all of the endospore-forming bacteria (Atibalentja 1960), who had examined this nematode parasite in some et al., 2004a, 2004b; Chen et al., 1997b; Ebert et al., 1996, 2004; detail, questioned this placement. It was not until the nematode Giblin-Davis et al., 2003a, 2003b; Metchnikoff, 1888; Sayre and parasite was re-examined using electron microscopy that its true Starr, 1985). affinities to bacteria rather than to protozoa were recognized Recent molecular work with 16S rRNA gene sequences sup- and the name Bacillus penetrans (Thorne, 1940) Mankau (1975) port Pasteuria as a monophyletic clade comprising well sup- was applied to it (Imbriani and Mankau, 1977; Mankau, 1975). ported lineages (Anderson et al., 1999; Atibalentja and Noel,

TABLE 55. Common characteristics of Pasteuria ramosa Metchnikoff (1888), Pasteuria penetrans sensu stricto emend. Starr and Sayre (1988a), Pas- teuria thornei Starr and Sayre (1988a), Sayre, Wergin, Schmidt and Starr (1991) emend. Noel, Atibalentja and Domier (2005), and “Candidatus Pasteuria usgae” Giblin-Davis, Williams, Bekal, Dickson, Brito, Becker and Preston (2003b) Characterisitic Description Morphological similarities as observed by light microscopy: Vegetative cells Microcolonies consist of dichotomously branched mycelium. Diameter of mycelial filaments similar. Mycelial filaments are seen in host tissues only during early stages of infection. Daughter microcolonies seem to be formed by lysis of “sacrifi- cial” intercalary cells. Nearly all vegetative mycelium eventually lyses, leaving only sporangia and endospores. Endospores Terminal hyphae or peripheral cells of the colony elongate and swell, giving rise to sporangia. A single endospore is produced within each sporangium. Endospores are in the same general size range. Refractivity of endospores, as observed on the light microscope, increases with maturity. Staining reaction Gram-positive Ultrastructural similarities: Vegetative cells Mycelial cell walls are typical of Gram-positive bacteria. Mycelial filaments divided by septa. Double-layered cell walls. Where they occur, mesosomes are similar in appearance and seem to be associated with division and septum formation. Endospores Typical endogenous spore formation. Identical sequences in endospore forma- tion: (a) septa form within sporangia; (b) sporangium cytoplast condenses to form forespore; (c) endospore walls form; (d) final endospore matures; and (e) “light” areas adjacent to endospore give rise to extrasporal fibers. Similar sequences of life stages Microcolonies. Fragmentation of microcolonies. Quartets of sporangia. Doublets of sporangia. Single sporangia. Free endospores. Host–bacterium relationships All parasitize invertebrates. Colonies first observed in the host are sedentary and located in the host’s musculature. Growth in muscle tissue eventually leads to fragmentation and entry of microcolonies into the coelom or pseudocoelom of the respective host. Microcolonies carried passively by body fluids. Colonization of hemolymph or pseudocoelomic fluid by the parasite is extensive. Host ranges are very narrow: Pasteuria ramosa occurs only in cladoceran water fleas, Pasteuria penetrans sensu stricto in the root-knot nematode Meloidogyne incognita, Pasteuria thor- nei in the lesion nematode Pratylenchus brachyurus, Pasteuria nishizawae in and Globodera, and “Candidatus Pasteuria usgae” in Belonolaimus. Host is completely utilized by the bacteria; in the end, the host becomes little more than a bag of bacterial endospores. Survival mechanisms Survive in field soil and at bottom of ponds. Resist desiccation. Moderately to strongly resistant to heat.

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sequences has been updated to depict the close relationship of Pasteuria spp. to other genera of the (Figure 38). Comparison of spoIIAB gene sequences amplified by PCR from DNA from different isolates (biotypes) of Pasteuria pen- etrans identified single nucleotide polymorphisms or SNPs, indicating genetic heterogeneity within populations obtained from both individuals, as well as populations of Meloidogyne spp. (Nong et al., 2007). DNA sequences from three different loci derived from a single-spore isolate of Pasteuria penetrans were identical, supporting the need for clonal populations for defini- tive studies on host preference (Trotter et al., 2004). To clarify the characteristics of the genus Pasteuria, the meanings of the terms “endospore” and “sporangium” must be modified slightly from their usual definition in order to be applicable to this genus. Metchnikoff observed the several stages of endosporogenesis that occurred in Pasteuria ramosa. In his discussion, he noted within each sporangium a single refractile body that stained with difficulty; he called this struc- ture, as we do today, an endospore. The Pasteuria endospore is not entirely typical of those found in Bacillus or Clostridium.

FIGURE 37. Drawings of Pasteuria penetrans sensu stricto (left) based FIGURE 38. Phylogenetic position of Pasteuria spp. inferred from on electron micrographs are compared with those of Pasteuria ramosa maximum-parsimony analysis of 16S rRNA gene sequences. The acces- (right) as drawn by Metchnikoff (1888). Starting at the top of the left sion numbers of the Pasteuria sequences used were as follows: “Pasteu- column is a vegetative colony of Pasteuria penetrans followed by daughter ria goettingianae”, AF515699; “Pasteuria hartismerei”, AJ878853; Pasteuria colonies, quartets of sporangia, doublets of sporangia, single sporangia nishizawae, AF134868; Pasteuria penetrans, AF077672; Pasteuria sp. para- and, finally, at the bottom the mature endospore within the old sporan- sitic on nematodes of the family Plectidae, AY652776; Pasteuria ramosa, gial wall. The drawings of Pasteuria ramosa on the right are placed in U34688; and “Candidatus Pasteuria usgae”, AF254387. The accession order of their occurrence in the life cycle of the parasite as reported by numbers for the other sequences were as in Atibalentja et al. (2000). Metchnikoff (1888). (Reproduced by permission from R.M. Sayre, W.P. Anacystis nidulans (Cyanobacteria) and Escherichia coli (Proteobacteria) were Wergin and R.E. Davis. (1977). Can. J. Microbiol. 23: 1573–1579). used as outgroup taxa. Bootstrap proportions (10,000 replications) are shown, wherever possible, for nodes that are relevant for Pasteuria spp. The figures not shown are 98, 96, 53, 87, and 93, respectively, for 2008; Atibalentja et al., 2000; Bekal et al., 2001; Bishop et al., branches leading to Pasteuria sp. on Plectidae, “Pasteuria hartismerei”, 2007; Ebert et al., 1996; Giblin-Davis et al., 2003b; Preston et al., “Pasteuria goettingianae”, Pasteuria nishizawae, and Pasteuria penetrans. Bootstrap support was 100% for each of the branches leading to Ali- 2003; Sturhan et al., 2005). Further genomic comparisons have cyclobacillus cycloheptanicus and Alicyclobacillus acidoterristris. The align- been made through the sequences of sporulation and other ment of the 16S rRNA gene sequences was performed with CLUSTAL X genes in different isolates of Pasteuria penetrans and Pasteuria (Thompson et al., 1997) and phylogenetic analyses were conducted ramosa (Bird et al., 2003; Charles et al., 2005; Preston et al., with PAUP* 4.0b10 (Swofford, 2003). “Pasteuria goettingianae”, “Pasteuria 2003; Schmidt et al., 2004; Trotter and Bishop, 2003). A phylo- hartismerei” and “Candidatus Pasteuria usgae” have not been published genetic tree (Atibalentja and Noel, 2008) comparing 16S rRNA as valid taxa. From Atibalentja and Noel (2008).

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[Au1] FIGURE 39. Scanning electron micrograph of endospores of Pasteuria penetrans sensu stricto attached to a juvenile of a root-knot nematode that has partially penetrated a tomato root. The endospores carried on the juvenile will germinate inside the plant and penetrate the developing nematode, completing their life cycle in their host. On decay of the FIGURE 40. Scanning electron micrograph of an endospore of Pasteu- plant root in soil, the endospores developed within the parasitized ria penetrans sensu stricto on cuticle of a second-stage juvenile of Meloid- nematode are released. Bar = 10 µm. ogyne incognita. This endospore has retained its exosporium, resulting in the appearance of a crinkled surface. Bar = 10 µm.

For one thing, the Pasteuria endospore has a mass of fibrous outgrowths emanating from the central body or core. These the endospore proper, and explicitly exclude the parasporal microfibrillar strands (usually called parasporal fibers, periph- fibers. Measurements and descriptions of parasporal fibers are eral fibers, or perisporium), which surround the central body of presented separately. the endospore, are structures comprised of glycoproteins that The attachment of endospores to their invertebrate hosts are presumed to function as adhesins involved in attachment of is mediated by the adhesins in their parasporal fibers. The the endospore to its invertebrate host (Figure 40) (Brito et al., attached endospore is often overlaid by seemingly nonfunc- 2004; Davies et al., 1994); (Persidis et al., 1991; Preston et al., tional remnants of the old sporangium (Figure 40). The pres- 2003; Schmidt et al., 2004). A monoclonal antibody that recog- ence or absence of these sporangial remnants may be due in nizes a glycan-containing epitope associated with adhesins on part to the length of time the sporangium has been subjected the surfaces of endospores of Pasteuria penetrans (Brito et al., to degradative processes in the soil or the amount of abrasion 2003; Schmidt et al., 2003) detects this epitope in extracts of received as the nematode host moves through its environs endospores from other species of infected nematodes (Preston ( Figure 41). When such sporangial material was significant, it et al., 2003), as well as Pasteuria ramosa (Schmidt et al., 2008). was included in the measurements of the endospores reported The epitope was not detected in extracts of endospores obtained herein. Even though sporangial material is sometimes seen, we from a number of Bacillus spp. that were tested, but is common have adopted the convention of calling the infectious unit on to endospores of all Pasteuria spp. that have been evaluated. the nematode exterior cuticle surface an endospore. Pasteuria Albeit an integral part of the endospore, the parasporal fibers endospores, at least in the case of the nematode parasites, also are arrayed differently in the different Pasteuria spp. discussed differ from those of Bacillus in that the former produce a germ herein. It is difficult to include these somewhat amorphous tube that penetrates the nematode cuticle and initiates bacte- fibrous masses in any precise measurements of endospores. For rial colonization within the nematode body. this reason, the measurements reported herein include only The type-descriptive material (Sayre and Starr, 1985) of Pas- the major and minor axes of the central body of the endospore, teuria penetrans (ex Thorne (1940) Sayre and Starr (1985) refers

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Validation List no. 20; (Sayre and Starr, 1986)], Pasteuria thornei [Sayre et al. (1988) and Starr and Sayre (1988a) description and illustrations serving as type; Validation List no. 26; (Starr and Sayre, 1988b)], and Pasteuria nishizawae [Sayre et al. (1991) as emended by Noel et al. (2005) description and illustrations serving as type; Validation List no. 41; (Sayre et al., 1992)]. We concur with the proposal of Ebert et al. (1996) to accept the Daphnia parasite that they isolated and studied, and whose 16S rRNA gene has been sequenced as the neotype for Pasteuria ramosa and the genus Pasteuria. Unfortunately, 16S rRNA gene sequence data are not available for Pasteuria thornei and this spe- cies of Pasteuria must be rediscovered before a more complete characterization can be made. Enrichment and isolation procedures Attempts to devise methods for in vitro cultivation of Pasteuria penetrans using defined media have proven difficult, but some success has been reported (Bishop and Ellar, 1991; Hewlett et al., 2006). The cultivation of Pasteuria penetrans outside of its nematode host has been reported by an independent commer- cial enterprise, but complexities and undisclosed composition of the media used have precluded their application and confir- mation in other laboratories. The distinctive morphology and unique relationship to invertebrate hosts shared by Pasteuria spp. (see Table 55) suggest that this commonality may be the harbinger of similarities in their physiological requirements for endospore germination, vegetative growth, sporulation, and their eventual axenic cultivation. Based on microscopic obser- vations, the physiological and physical requirements for their FIGURE 41. Endospore of Pasteuria penetrans sensu stricto attached to the growth in vivo would appear to be similar. Summarized briefly, cuticle along the lateral field of the juvenile of a root-knot nematode. vegetative growth of Pasteuria spp. seems linked through the The exosporium has been sloughed, exposing the central dome of the environment provided in the coelom/pseudocoelom of their endospore; the peripheral fibers can be distinguished. Bar = 0.5 µm. different invertebrate hosts. The hemolymph of the cladoceran or the pseudocoelomic fluid of the nematode allows for the to the bacteria occurring on the root-knot nematode Meloid- exchange of nutrients and waste products. Also, the coelom/ ogyne incognita. Hence, the name Pasteuria penetrans must refer pseudocoelom provides space for mycelial colony development. to that organism (Starr and Sayre, 1988a). This morphotype, These colonies fragment after they reach a critical size. Finally, however, has been isolated from other Meloidogyne spp. and it is reasoned that various factors, possibly the accumulation of comprises several genotypes that may defy or challenge easy bacterial biomass and metabolites, as well as the onset of senility species definition (Sturhan et al., 2005). Some other observed in the invertebrate hosts, trigger sporulation of the bacteria. members of Pasteuria are demonstrably different from Pasteuria The similar physical conditions found in the separate host penetrans using a Linnaean species concept (typological) with species suggests that the bacteria might have common nutri- molecular corroboration and should be assigned to other taxa. tional requirements; hence, when the requirements for axenic The first such assignment was made for the bacterium from cultivation of one Pasteuria sp. become known, they may, with the lesion nematode Pratylenchus brachyurus, to which the name slight modification, apply to other species. The unique host Pasteuria thornei Starr and Sayre (1988a) was affixed (Starr and specificities of different Candidatus species (Giblin-Davis et al., Sayre, 1988a). Because the obligate endoparasitic nature of 2004; Giblin-Davis et al., 2003a; Sturhan et al., 2005), as well as Pasteuria currently prevents cultivation of axenic type strains, biotypes of Pasteuria penetrans defined by a marked preference “Candidatus” status has been proposed for each new provisional for different Meloidogyne spp. (Oostendorp et al., 1990; Stirling, species designation in this genus (Giblin-Davis et al., 2003b; 1985), supports a role for the host in conferring virulence and Murray and Stackebrandt, 1995; Murray and Schleifer, 1994; host specificity. The host may play an active role in the matu- Stackebrandt et al., 2002). All of the previously named spe- ration of endospores and endow it with adhesins that serve as cies in the genus Pasteuria Metchnikoff (1888) (Skerman et al., virulence factors through their recognition of receptors on the 1980) have nomenclatural standing and remain as species with cuticle of the host and the attachment that is required for infec- validly published names. tion (Preston et al., 2003). The species that remain valid include Pasteuria ramosa Since isolation and cultivation of Pasteuria ramosa, apart from Metchnikoff (1888) [Skerman et al. (1980) as emended by Starr in its cladoceran host, has not yet been achieved, this bacterium et al. (1983) serving as type; see Judicial Commission (1986), is usually studied in field samples of the infected invertebrate Wayne (1986)], Pasteuria penetrans (ex Thorne, 1940) [Sayre host. The following procedures may increase the chances of and Starr (1985) description and illustrations serving as type; finding Pasteuria ramosa in its natural habitat.

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1. Cladocerans should be collected during the warmest part the number of endospores per gram of soil and the extent of the growing season. The parasite is most often found in to which soils are suppressive for plant nematodes (Chen Daphnia magna, but also in other Daphnia spp. (Ebert, 2005). and Dickson, 1998; Oostendorp et al., 1990). Also, meth- 2. Because the frequency of occurrence of the parasite in a ods have been developed for the quantification of Pasteuria cladoceran population may be low (less than 10%), a large endospore concentrations in tomato root material (Chen number of living specimens needs to be examined to increase et al., 1996), the extraction and purification of endospores the odds for detection. Prevalence, however, may reach up to (Chen et al., 2000), and for determining suppressive soils 100% of all the adult hosts (Duncan and Little, 2007). caused by Pasteuria (Chen et al., 1997a; Dickson et al., 1994; 3. The internal parasites are most easily identified by using an Stirling, 1984; Walia et al., 2004). PCR-based methods have inverted microscope at magnifications of ×100–250. Infected been developed for amplifying and sequencing 16S rRNA Daphnia are typically large and much less transparent than genes from soil samples and single nematodes (Atibalentja uninfected animals. The parasite fills the entire body cavity. et al., 2004b; Duan et al., 2003). Immunoassays using poly- Although axenic cultivation of Pasteuria penetrans has been clonal (Costa et al., 2006; Fould et al., 2001) and monoclonal reported but not confirmed, investigations have been limited to (Schmidt et al., 2003) antibodies directed against endospore studies on naturally or artificially infected nematode hosts (Bekal surface proteins have been developed for the detection and et al., 1999; Chen et al., 1997a; Chen and Dickson, 1998; Imbri- quantification of bacterial endospores in soil and tissue sam- ani and Mankau, 1977; Mankau, 1975; Mankau and Imbriani, ples. PCR-based assays for sporulation and other genes have 1975; Sayre et al., 1983, 1988; Sayre and Wergin, 1977; Starr and been developed for the identification and quantification of Sayre, 1988a) and exploration of the bacterium’s potential as a the vegetative cells in plant tissues (Schmidt et al., 2004). biological control agent against plant-parasitic nematode popula- 4. Occurrence of endospores on the surfaces of nematodes is tions (Chen and Dickson, 1998). Consequently, the studies have most easily observed by means of an inverted microscope depended on finding, maintaining, and manipulating nematode at ×250–400 magnification. Several stains, e.g., crystal vio- populations infected with these bacteria. Because of this direct let, cotton blue, Brilliant Blue G, etc., are useful for visual- dependence on host–nematode populations, the procedures izing external spores. The endospores also may be readily and methods used for maintaining members of the nematode- identified with a fluorescent immunoassay and monoclonal associated Pasteuria are by-and-large those used in maintaining antibodies that recognize adhesin-related epitopes (Davies the nematodes (Southey, 1986; Zuckerman et al., 1984). et al., 1994; Schmidt et al., 2003). The application of immu- A few generalizations about Pasteuria spp. follow: noassays avoids potential misidentification of endospores of Paenibacillus spp. that may be associated with entomopatho- 1. The group of nematodes that are parasitized by Pasteuria spp. genic rhabditid nematodes (El-Borai et al., 2005; Enright is widespread and diverse. The bacterial parasite has been et al., 2003). reported from about 116 nematode genera and 323 nema- 5. Direct confirmation of the presence of members of the Pas- tode species. Pasteuria spp. have been reported in a dozen teuria group inside the nematode can be made by micro- states of the United States, as well as roughly 51 countries on scopic examination of the pseudocoelom of nematodes, also five continents and on various islands in the Atlantic, Pacific, at ×250–400 magnification. Both juveniles and adults should and Indian Oceans (Chen and Dickson, 1998; Ciancio et al., be examined for the characteristic mycelial colonies and 1994; Sayre and Starr, 1988; Sturhan, 1988). endospores. Treating nematodes with methyl blue and lac- 2. Members of nematode-associated Pasteuria will most likely tophenol stain works well for visualizing the various develop- be found in soils where nematode populations have been mental stages within the nematode pseudocoelom (Serracin consistently high and are causing crop damage (in the case et al., 1997). In uninfected nematodes, the anterior region of plant-parasitic nematodes only). However, numerous of the esophagus is clear, with internal structures visible; nematode suppressive soils have been identified where Pas- when filled with endospores, the region will not be clear and teuria causes a precipitous drop in plant nematode numbers internal structures will be masked by the bacterium. (Chen and Dickson, 1998). Planting of susceptible crops 6. Detection of the bacterium in the sedentary endoparasitic is necessary for maintenance of the nematode populations nematodes depends on manual (Thorne, 1940) and/or and multiplication of Pasteuria spp. Pasteuria spp. may also be enzymic (Dickson et al., 1970) removal of root-tissues from associated with plant nematodes in greenhouse situations. around the female nematodes. The freed females are placed 3. To find the bacterial endospores, nematodes are extracted on glass slides, crushed, and their body contents are exam- from the suspected soil, e.g., by a centrifugal-flotation ined microscopically for vegetative stages and endospores of method (Jenkins, 1964). Increasing the sucrose concentra- the bacterium. tion used for extraction of nematodes results in a higher percentage recovery of endospore-filled specimens (Oost- Maintenance procedures endorp et al., 1991). Other separation methods, relying on the nematode’s mobility (e.g., Baermann, 1917), may not Since the recently reported method of axenic cultivation is yield those nematodes that are heavily encumbered with proprietary and has not been optimized or confirmed, Pasteu- endospores or endospore-filled bodies since such nematodes ria spp. are maintained by co-cultivation with their respective are partially immobilized. Addition of healthy nematodes to invertebrate host. soils, together with subsequent extraction and examination, Pasteuria ramosa can be grown in the laboratory in clonal is the most commonly used bioassay for determining the cultures of Daphnia magna. (Ebert et al., 2004). An endospore- presence of members of the genus Pasteuria in soil. Attach- filled body of Daphnia magna is shown in Figure 42. Daphnia ment assays have been developed that allow estimations of are maintained on a diet of chemostat-grown unicellular green

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FIGURE 43. Section through a germinated endospore of Pasteuria pen- etrans sensu stricto; the penetrating germ tube follows a sinuous path as it travels the cuticle and hypodermis of the nematode. Bar = 0.5 µm.

FIGURE 42. Photomicrograph of Daphnia magna showing body cavity filled with endospores of Pasteuria ramosa.

algae Scenedesmus sp. At 20°C, an infected Daphnia magna pro- duces several million Pasteuria spores within about 40 d. Endospores used to inoculate healthy Daphnia magna are obtained from two sources: (a) the crushed bodies of living or dead parasitized cladocerans in late stage infections; and (b) sedi- ments from the bottoms of aquaria (or ponds) in which dead and parasitized cladocerans had accumulated. Frozen cadavers can also be used. The infection rate is dose-dependent. High infec- tion rates can be reached by adding 10,000 endospores to jars with 20 ml water and a single Daphnia magna in each jar (Regoes et al., 2003). Resistance of Daphnia magna clones to Pasteuria FIGURE 44. Section of a mycelial colony of Pasteuria penetrans sensu ramosa is widespread (Carius et al., 2001; Decaestecker et al., 2003; stricto in the pseudocoelom of the nematode. The septate hyphae Little and Ebert, 1999) and may be the most common reason for appear to bifurcate at the margins of the colony. Bar = 0.5 µm. a failure to cultivate the bacterium. Furthermore, there are strong host clone–parasite isolate interactions (Carius et al., 2001). Nematode-associated Pasteuria can be maintained in a system consisting of the immediate nematode host and its host plant. A good example is the system consisting of Pasteuria penetrans– Meloidogyne incognita and tomato plants (Sayre and Wergin, 1977). To initiate and increase a bacterial population, dried bac- terial endospore preparations (e.g., Stirling and Wachtel, 1980) are mixed into soils that are heavily infested with juveniles of Meloidogyne incognita. The juveniles become encumbered with the bacterial endospores as they move through the soil in a random fashion (Figure 39). The endospores adhering to the nematode cuticle are carried by the juvenile into tomato roots, where germination occurs after the nematode initiates feeding. An endospore penetrates the cuticle by means of a single germ tube (Figure 43), which extends into the pseudocoelomic cavity. The bacterium then enters into its vegetative endoparasitic developmental stages (Figure 44 and Figure 45). Finally, bacte- ria form in the mature and moribund host nematodes and in FIGURE 45. Hyphal cells of Pasteuria penetrans sensu stricto are bounded sporangia that contain endospores (Figure 46, Figure 47, and by a compound wall consisting of a double membrane. A mesosome is Figure 48). In summary, the developmental stages of the bacterium associated with the septum. Bar = 0.5 µm.

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FIGURE 48. Median section through a sporangium of Pasteuria pen- etrans sensu stricto containing a fully mature endospore. Final stages of endospore differentiation include formation of an encircling mem- brane or exosporium and emergence of parasporal fibers within the granular material that lies laterally around the spore. Bar = 0.5 µm.

include recognition and attachment to a susceptible host nema- tode, infection of the host nematode via a germ tube, followed by vegetative growth, sporulation, and maturation within the host pseudocoelom. FIGURE 46. An early stage of endospore development in Pasteuria pene- Endospores of Pasteuria penetrans growing inside Meloidogyne trans sensu stricto is shown in this median section through a sporangium. incognita can be harvested by two methods. The simplest pro- An electron-opaque body has formed with the forespore; the body is cedure is to allow the nematode-infested plant roots to decay surrounded by membranes that will condense and contribute to the in situ in soil; during such decay, about 2 × 106 endospores are multilayered wall of the mature endospore. Bar = 0.5 µm. released from each female nematode. The soil containing the endospores is air-dried, mixed, and stored. Such preparations have yielded bacterial endospores that can attach to the juve- nile of their respective host nematode even after several years in storage (Mankau, 1973). A second method for obtaining a more concentrated preparation of the bacterial endospores has been demonstrated (Stirling and Wachtel, 1980). Freshly hatched juveniles of Meloidogyne incognita may be encumbered with endospores by placing them in aqueous suspensions con- taining endospores. A centrifuge method can be used to help obtain consistent attachment of endospores to host nematodes (Hewlett and Dickson, 1993). These encumbered juveniles are then allowed to penetrate roots of tomato seedlings. After the life cycle of the nematode is completed in soil, the galled roots are harvested, washed, air-dried, ground into a fine powder, and stored. Such preparations have provided adequate sources of endospores for use in bioassays and other procedures. A more efficient and rapid method of obtaining endospore-filled female nematodes is by using an enzyme (cellulase and/or pectinase) preparation (Brito et al., 2003; Chen et al., 2000; Schmidt et al., 2004). Purification can be achieved by selective filtration steps and centrifugation in sucrose or renografin (sodium diatrizo- ate) gradients (Chen et al., 2000). Pasteuria thornei and “Candidatus Pasteuria usgae” can be maintained in similar systems, except that the bacteria must be maintained on the migratory endoparasitic host nematode Pasteuria brachyurus or the ectoparasitic nematode Belonolaimus longicaudatus, respectively. Pasteuria brachyurus may be collected FIGURE 47. Section through a sporangium of Pasteuria penetrans sensu from roots of infected hosts, whereas Bacillus longicaudatus may stricto with an almost mature endospore. The lateral regions (light be collected from around roots of infected plants. Numerous areas) will differentiate into parasporal fibers. Bar = 0.5 µm. bacterial endospores are liberated upon decay of the plant

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roots and the cadavers of infected nematodes. Healthy juveniles (1983), have been recommended (Starr et al., 1983) and or adults of either nematode migrating through such soils can approved (Judicial-Commission, 1986). The detailed drawings, become encumbered with endospores of their respective bacte- photomicrographs, and lengthy description offered by Metch- rial parasite and repeat the developmental cycle, with not only nikoff (1888) provided a sound basis for comparing his species maintenance of the bacterium, but also a net increase in the with the current cladoceran parasites. As stated above, a Pas- endospore content of the soil. teuria ramosa-like strain was discovered infecting Moina rectiro- At present, the only method of producing endospores of Pas- stris (Sayre et al., 1979) that was used in the emendation of the teuria nishizawae is to collect infected females and cysts. This is species (Starr et al., 1983). We support the proposal of Ebert difficult due to the fragile nature of infected cysts and the dif- et al. (1996) that the Daphnia-parasitic Pasteuria ramosa charac- ficulty in identifying them. At a certain stage, the infected cysts terized from the same host as Metchnikoff (1888) from Europe are usually a grayish-green color, but this coloration is often dif- be designated the neotype for Pasteuria ramosa Metchnikoff ficult to observe. Infected females cannot be readily identified (1888) and that the Moina isolate of Sayre et al. (1979) be com- without crushing them. pared directly to the neotype in future studies. A number of investigators have cultivated members of Pasteu- One reason investigators (Henrici and Johnson, 1935; Hirsch, ria in three-membered systems consisting of plant-tissue cultures, 1972) have been interested in re-examining Pasteuria ramosa gnotobiotically reared nematodes, and the desired bacterium stems from the questions raised by Metchnikoff’s assertion that free of contaminating microbes (Bekal et al., 1999; Chen and the bacterium divides longitudinally. Metchnikoff suggested Dickson, 1998). Once perfected, these cultural methods are use- that cells of Pasteuria ramosa undergo a longitudinal fission, giv- ful for maintenance of these bacteria, at least until methods for ing rise to a branched structure in which the two daughter cells their axenic cultivation become generally available. remain attached at their tips. Based on a hypothesis about the evolution of division patterns in bacteria, he concluded that Differentiation of the genus Pasteuria from other genera Pasteuria ramosa, a fairly primitive bacterium in his view, divided Table 55 summarizes the characteristic features of species of the longitudinally. genus Pasteuria. Observations on the Pasteuria ramosa-like parasite of Moina (Sayre et al., 1979) indicated that cleavage indeed occurs in Taxonomic comments three planes, as evidenced by the spherical mycelial colonies. De Toni and Revisan (1889) provided the first generic diagnosis However, no common plane of division was found, as illustrated of Pasteuria; it followed quickly and closely the original descrip- in drawing 2 of the Metchnikoff (1888) paper, starting at the tion of Pasteuria ramosa by Metchnikoff (1888). However, some surface of the cauliflower-like growth and ending at its interior. other early investigators, particularly those interested in taxo- The prominent bifurcations in the mycelium (Figure 49), which nomic coherence, not having observed this enigmatic organism Metchnikoff also observed and which may have prompted his and relying solely on descriptions, rejected both the generic longitudinal fission theory, are not products of atypical fission and specific concepts (Lehmann and Neumann, 1896; Migula, but rather are probably the branched distal or terminal cells 1904). Laurent (1890) suggested that a bacteroid-forming spe- of the mycelium undergoing rapid enlargement during forma- cies from nodules on leguminous plants, together with Pasteu- tion of endogenous spores. These terminal cells develop into ria ramosa, comprised the new family he erected, Pasteuriaceae. sporangia. Similarly, Vuillemin (1913) believed that a generic relationship Of some historical interest are a few scattered reports in existed between Nocardia and Pasteuria. De Toni and Revisan the literature about organisms similar in appearance to Pas- (1889) speculated that Pasteuria ramosa, because of its ability to teuria ramosa. In these reports, each author came to a differ- form endospores, should be placed in the subtribe Pasteurieae of ent taxonomic decision about the organism, as follows: Torula the tribe Bacilleae. The unusual morphology of Pasteuria ramosa became the basis for numerous suppositions about its affinities to other bacterial groups (Buchanan, 1925). This speculative process continued up until recently (Sayre et al., 1983; Sayre and Starr, 1985, 1988; Starr and Sayre, 1988a). The advent of 16S rRNA gene analyses indicated that Pasteuria is a deep lin- eage within the (Atibalentja et al., 2000; Ebert et al., 1996). Although it has been classified within the “Alicyclobacil- laceae” (Garrity et al., 2005), the low sequence similarity of 85% and its distinctive phenotype suggests that it would be more properly classified within its own family, the Pasteuriaceae. A significant change made since the 9th edition of the Man- ual is the reduction in the confusion between Metchnikoff’s Gram-positive, endospore-forming, mycelial cladoceran para- site with certain Gram-negative, budding, non-prosthecately appendaged aquatic bacteria. Conservation of Pasteuria ramosa sensu Metchnikoff (1888) on the basis of type-descriptive mate- rial, as well as rejection of ATCC 27377 as the type of Pasteuria ramosa Metchnikoff (1888) because it actually is a quite dif- FIGURE 49. Cross-section of a fragmenting mycelium of Pasteuria ferent organism (Planctomyces staleyi Starr, Sayre and Schmidt ramosa in the body cavity of a cladoceran. Bar = 2.0 µm.

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or other yeast species (Ruehberg, 1933); two different genera are summarized elsewhere (Sayre and Starr, 1988; Sayre et al., of the microsporidia (Jirovec, 1939; Weiser, 1943), and a hap- 1988; Starr and Sayre, 1988a). losporidium (Dermocystidium daphniae Jirovec (1939) and a pos- Acknowledgements sible intermediate stage in the life cycle of a Dermocystidium sp. (Sterba and Naumann, 1970). These results probably stem from This chapter is dedicated to the memory of Dr Richard Sayre the inability of these investigators to cultivate the particular and his wife, Diane, who perished on 10 June 1998 in a boating organism they observed, taken together with their dependence accident in the Galapagos Islands. solely on morphology and staining reactions as the basis for We thank R. E. Davis for calling our attention to the genus Pas- their descriptions and classifications. Surprisingly, Metchnikoff teuria and for his help in initiating its study. Thanks also go to W. P. (1888), the first person to report on Pasteuria, recognized it cor- Wergin, J. R. Adams, C. Pooley, R. Reise, and S. Ochs for sustained rectly as a bacterium, whereas the later workers did not and, technical advice and assistance, particularly in the preparation of moreover, were apparently unaware of Metchnikoff’s work. the figures involving transmission and scanning electron micros- Until rather recently, taxonomic studies of Pasteuria were car- copy. Thanks are due R. L. Gherna for supplying strain ATCC ried out mainly by nematologists. Cobb (1906) erroneously des- 27377. We are grateful to J. M. Schmidt for her corroboration dur- ignated such microbes as protozoan parasites of nematodes. At ing the period when the taxonomic confusion between Pasteuria first glance, it would appear from the literature that subsequent and Planctomyces was being corrected. Skillful bibliographic and workers (Steiner, 1938; Thorne, 1940) had independently come redactional assistance was provided by P. B. Starr. to the same conclusion. However, their conclusions were prob- Further reading ably by consensus. Members of Pasteuria were then, and largely still are, essentially known only to the community of plant nem- Chen, Z.X. and D.W. Dickson. 2004. Biological control of nematodes with atologists. When Thorne described Duboscqia penetrans as a pro- bacterial antagonists. In Chen, Chen, and Dickson (Editors), Nematol- tozoan, he could not have realized its bacterial nature because ogy Advances and Perspectives, Vol. 2, Nematode Management and electron microscopes were not then available. Later, Williams Utilization, CABI Publishing, Cambridge, MA, pp. 1041–1082. Gowen, S., K.G. Davies and B. Pembroke. 2008. Potential use of Pasteu- (1960) studied a similar organism in a population of root-knot ria spp. in the management of plant parasitic nematodes. In Ciancio nematodes from sugarcane, presented an interpretation of its and Mukerji (Editors), Integrated Management and Biocontrol of life stages, and indicated some reservations about Thorne’s Vegetables and Grain Crops Nematodes, Springer, Dordrecht, The identification. Canning (1973) also doubted the identification Netherlands, pp. 205–219. as a protozoan and stressed the organism’s fungal characteristics. Finally, Mankau (1975) and Imbriani and Mankau (1977) estab- Differentiation and characteristics of the species lished the bacterial nature of the organism and brought its and Candidatus species of the genus Pasteuria attendant taxonomic problems to the attention of bacteriolo- The differential characteristics of nominal Pasteuria and Candi- gists. Some results of the ensuing interdisciplinary enterprise datus species are given in Table 56.

List of species of the genus Pasteuria 1. Pasteuria ramosa Metchnikoff 1888, 166AL [Nom. Cons. DNA G+C content (mol%): not reported. Opin. 61 Jud. Comm. 1986, 119. Not Pasteuria ramosa in the Type strain: descriptions and illustrations serving as type. sense of Henrici and Johnson 1935, Hirsch 1972, and Sta- GenBank accession number (16S rRNA gene): AY762091 (not type ley 1973; see Starr et al. (1983) and Judicial Commission material). (1986)] ra.mo’sa. L. adj. ramosus much-branched. 2. Pasteuria nishizawae Sayre, Wergin, Schmidt and Starr VP Gram-positive. Sporangia and microcolonies are parasitic in 1992, 327 (Effective publication: Sayre, Wergin, Schmidt the hemocoel of cladocerans, water fleas of the genera Daphnia and Starr 1991, 562.) (emend. Noel, Atibalentja and Domier and Moina. Usually occur attached to one another at pointed 2005, 1683.) ends of the teardrop-shaped sporangia, forming quartet, trip- ni.shi.za’wae. M.L. gen. n. nishizawae of Nishizawa, named after let, and doublet configurations. The rounded end of the spo- Tsutomu Nishizawa, a Japanese nematologist who discovered and rangium encloses a single refractile endospore, having axes first investigated bacterial parasites of cyst-forming nematodes. of 1.37–1.61 × 1.20–1.46 µm, narrowly elliptic in cross-section. Gram-positive vegetative cells, forms endospores. Obligate Endospores are 4.2–5.4 × 4.9–6.0 µm. Nonmotile. Endospores endoparasitic bacterium of the pseudocoelom of Heterodera are resistant to desiccation, but with only limited heat tolerance. glycines (). Microcolony shape is cauli- They have been recovered from 30-year-old pond sediments flower-like initially and later fragments into clusters of elongated (Decaestecker et al., 2004). Vegetative stages are cauliflower- grape-like immature sporangia occurring in configurations of like, septate, mycelial growths that branch dichotomously and octets, quartets, and doublets. Sporangia are cup-shaped with fragment to form microcolonies. Has not been cultivated axeni- diameters and heights (under the light microscope) of 5.3 cally, but can be grown in the laboratory with the invertebrate and 4.3 µm, respectively, and (under the transmission electron host. The type-descriptive material consists of descriptions and microscope) of 4.4 and 3.1 µm, respectively. Sporangial wall and illustrations in Metchnikoff’s original publication (Metchnikoff, mother cell matrix disintegrate at maturity leaving the exosporium as 1888) and elsewhere (Ebert et al., 1996; Sayre et al., 1979, 1983; the outermost layer of the endospore. The surface of the exospo- Starr and Sayre, 1988a; Starr et al., 1983). rium is velutinous to hairy. The stem cell is observed occasionally.

BBergeys_Ch10.inddergeys_Ch10.indd 334848 22/19/2009/19/2009 7:50:007:50:00 PMPM GENUS I. PASTEURIA 349 (continued) P. usgae” P. 1.44–2.34 × P. penetrans P. to broadly elliptical in section expose inner endospore inward in the developed endospore; no clear external markings Candidatus Oblate spheroid, ellipsoid Basal cortical wall thins to Rarely seen 5. “ Basal portion collapses Same as 1.15–1.43 2.40–3.91 Pasteuria usgae” × ometimes almost spherical, narrowly elliptical in section expose inner endospore sporangium seen somewhat sublaterally sometimes collapsing at bases; no clear external markings cortical wall, bending sharply downward to form an attachment layer of numerous shorter fibers P. thornei P. Candidatus Small, elongate clusters Like cauliflower floret Oblate spheroid; ellipsoid, s Basal cortical wall thins to Surrounds endospores 4. Remains essentially rigid, Long fibers arise directly from , and “ Pasteuria thornei 1.30–1.54 0.96–1.20 × emend., elongated grapes broadly elliptical in section from thickened outer wall sublaterally in the developed endospore; no clear external markings cortical wall, gradually arching downward to form an attachment layer of numerous shorter fibers P. penetrans P. Spherical to cluster of Oblate spheroid, ellipsoid to Basal annular opening formed 3. Present PresentSurrounds endospore somewhat Present Basal portion collapses inward Stranded inclusions observed Stranded inclusions observed Fibers arise directly from but 1.2–1.4 0.99–1.21 P. penetrans P. × Pasteuria penetrans sensu stricto , initially, later fragments initially, into cluster of elongated grape-like sporangia to narrowly elliptical in section constant and is the depth of pore hairy surface not in basal laterally, or polar areas mother cell matrix of endospore disintegrate, leaving exosporium as the outermost layer of endospore; no clear external markings additional layer is formed on obverse surface of endospore sometimes seen P. nishizawae P. Seen occasionallyOblate spheroid, ellipsoid Seen occasionally Neither stem cell nor second 2. Sporangial wall and Stranded inclusions Same as Pasteuria nishizawae , 1.20–1.46 1.4–1.8 × sporangia ellipsoid, narrowly elliptic in section external markings divide sporangium in three parts stranded inclusions laterally from cortical wall, bending sharply downward secondarynumerous yield to fibers arrayed internally toward the granular matrix P. ramosa P. Pasteuria ramosa Vertical Horizontal Horizontal Horizontal Horizontal Not observed Surrounds endospore Teardrop2.12–2.773.40–4.35Remains rigidly in place; Cup 4.1–4.7 2.8–3.4 3.0–3.9 Cup 2.26–2.60 2.22–2.7 1.96–2.34 Rhomboidal Cup to rhomboidal 2.73–4.97 4.7–7.1 1.37–1.61 0.28–0.34–Long primary fibers arise 0.22–0.26 0.3±0.1 0.17–0.23 0.28±0.11 0.36–0.59 0.13±0.01 0.29±0.1 a a m µ a m a µ m a m µ m µ Comparison of µ

Shape Diameter, major axis to sporangium base wall Height, Fate of sporangial wall at maturity of endospore origin and orientation Sporangia Orientation of Cell dimensions, Diameter, Colony shape Like cauliflower floret Like cauliflower floret Stem cellCentral body Remains attached to most Oblate spheroid, an Pore Occurrence Characteristics Absent – Present Thickness of basal wall Present Present Present Characteristic 1. Exosporium Not observed Present, velutinous to Partial middle spore TABLE 56. TABLE Wall thickness, Wall Protoplast Contains pronounced Parasporal structures,

BBergeys_Ch10.inddergeys_Ch10.indd 334949 22/19/2009/19/2009 7:50:007:50:00 PMPM 350 FAMILY V. PASTEURIACEAE ) P. usgae” P. Belonolaimus P. penetrans P. longicaudatus tode cuticle by germ tube numbers on cuticular surface lature; no attachment to pseudocoelom walls seen adults Candidatus Nematodes ( Direct penetration of nema- Spores accumulate in large Same as Pseudocoelom and muscu- 5. “ 3rd, 4th stage juveniles and ) Pratylenchus brachyurus but not seen numbers on cuticular surface some strands are formed and partial collapse may occur musculature; no attachment to pseudocoelom walls seen juveniles and adult P. thornei P. Nematodes ( Direct penetration suspected Spores accumulate in large Persists, but more granular; Pseudocoelom and 4. Yes, in 2nd, 3rd, and 4th stage Yes, ined by phase-contrast light microscopy and scanning electron microscopy. b Meloidogyne ) occasionally seen in 2nd stage juvenile incognita cuticle by germ tube numbers on cuticular surface occurs; sporangial wall collapses; base is vacuolate musculature; no attachment to pseudocoelom walls seen P. penetrans P. Mostly only in female, Nematodes ( Direct penetration of nematode Spores accumulate in large 3. Becomes coarsely granular; lysis Pseudocoelom and ) Heterodera glycines Heterodera and cyst ( nematode cuticle by germ tube niles, rare on male strands are formed and partial collapse may occur lature; no attachment to pseudocoelom walls seen P. nishizawae P. Cyst nematodes Direct penetration of Spores accumulate on juve- 2. Persists, numerous Pseudocoelom and muscu- ) Moina , Daphnia ( to occur through gut wall attach or accumulate on surface of cladoceran material musculature; sometimes found attached to coelom walls P. ramosa P. – No, only in female Not known; suspected Spores not observed to (continued)

cycle in nematode juveniles of host spores on host Rarely seen in males but, when observed, thought to be sex-reversed males (Hatz and Dickson, 1992). Measurements are based on preparations examined by transmission electron microscopy. Somewhat different apparent sizes are obta Measurements are based on preparations examined by transmission electron microscopy. Completes life Source of host Pond mud, freshwater Soil, plants Soil, plants Soil, plants Soil, plants Host Cladocerans Mode of penetration Attachment of TABLE 56. TABLE CharacteristicMatrix, at maturity Persists as fine granular 1. a b Location in host Hemocoel and

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The central body is oblate spheroid, ellipsoid to narrowly ellip- and Starr and Sayre (1988a). Pasteuria penetrans differs from tical. Orientation of major axis to sporangium base is horizontal other described members of Pasteuria in host specificity, in size with diameter and height of 2.1 and 1.7 µm, respectively (when and shape of sporangia and endospores, and in other morpho- viewed under the light microscope) or 1.6 and 1.3 µm, respec- logical and developmental characteristics. tively (when viewed by transmission electron microscopy). The The influence of temperature on the development of Pas- epicortical layer entirely surrounds the cortex. A laminar inner teuria penetrans in Meloidogyne spp. has been observed in growth spore coat with alternating layers of dense and light materials chambers (Hatz and Dickson, 1992; Serracin et al., 1997; occurs between the outer spore membrane and the epicortical Stirling, 1981). The parasite’s greatest endospore attachment layer. The outer spore coat consists of several layers of electron- rate to second-stage juveniles was at 30°C and the bacterium dense materials and is surrounded laterally and ventrally by developed more quickly within its nematode host at 30 and microprojections. Including the microprojections, the outer 35°C than at 25°C. Development time quickly decreases as tem- spore coat is thickest at the top of the central body and then perature decreases, e.g., at 35, 28, and 21°C, mature endospores tapers gradually to 0.2 µm at the spore equator and to 0.1 µm or were detected at 28, 35, and >90 d, respectively (Hatz and Dick- less around a 0.3 µm wide basal pore. Parasporal structures con- son, 1992; Serracin et al., 1997). sist of long primary fibers arising laterally from the outer spore DNA G+C content (mol%): not reported. coat and bending downwards to yield numerous secondary Type strain: descriptions and illustrations serving as type. fibers arrayed ventrally. Depending upon the extent of invagi- GenBank accession number (16S rRNA gene): AF077672 and nation of the basal adhesion layer, additional partial hirsute AF375881 (not type material). layers may be present on the obverse face of the central body. 4. Pasteuria thornei Starr and Sayre 1988, 328VP (Effective Spores attach to second-stage juveniles in the soil, but rarely publication: Starr and Sayre 1988a, 28.) to males in the soil. The cuticle and body wall are penetrated by the germ tube that develops after the infective second-stage thor’ne.i. M.L. gen. n. thornei of Thorne, named after Gerald juvenile penetrates the host plant root. The life cycle is com- Thorne, a nematologist from the United States, who described pleted only in the pseudocoelom of females, but may also be and named this parasite of Pratylenchus as a protozoan parasite. completed in the cyst (female cadaver). The only confirmed Gram-positive vegetative cells. Mycelium is septate; hyphal host is Heterodera glycines. Attachment of endospores to second- strands, 0.2–0.5 µm in diameter, branch dichotomously. Spo- stage juveniles of Globodera rostochiensis () rangia, formed by expansion of hyphal tips, are rhomboidal with endospores obtained from Heterodera elachista (upland rice in shape, approximately 2.22–2.70 µm in diameter and 1.96– nematode), Heterodera lespedezae (lespedeza cyst nematode), Het- 2.34 µm in height. Each sporangium is divided into two almost erodera schachtii (sugarbeet cyst nematode), and Heterodera trifolii equal units. The smaller unit, proximal to the mycelium, is (clover cyst nematode) indicates that these nematode species not refractile and contains a granular matrix interspersed with may be hosts. Completion of the life cycle of Pasteuria nishizawae many fibrillar strands. The refractile apical unit is cone shaped; in these nematode species has not been confirmed. it encloses an ellipsoidal endospore, sometimes almost spherical, DNA G+C content (mol%): not reported. having axes of 0.96–1.20 × 1.15–1.43 µm, with cortical walls Type strain: descriptions and illustrations serving as type. about 0.13 µm in thickness. A sublateral epicortical wall gives the GenBank accession number (16S rRNA gene): AF134868 and endospore a somewhat triangular appearance in cross-section. AF516396. The tapering outer cortical wall at the base of the endospore forms an opening approximately 0.13 µm in diameter. Sporan- 3. Pasteuria penetrans (ex Thorne 1940) Sayre and Starr 1986, VP gia and endospores are found as parasites of lesion nematodes 355 (Effective publication: Sayre and Starr 1985, 163.) (Pratylenchus spp.). Has not been cultivated axenically; the type- (emend. Sayre, Starr, Golden, Wergin and Endo 1988, 28; descriptive material consists of the text and photographs in Duboscqia penetrans Thorne 1940, 51.) Starr and Sayre (1988a) and Sayre et al. (1988). Pasteuria thornei pen’e.trans. L. v. penetro pres. part. penetrans to enter. differs from Pasteuria penetrans and other members of Pasteuria Gram-positive vegetative cells. Mycelium is septate; hyphal in host specificity, size and shape of sporangia and endospores, strands, 0.2–0.5 µm in diameter, branch dichotomously. The and other morphological and developmental traits. sporangia, formed by expansion of hyphal tips, are cup shaped, DNA G+C content (mol%): not reported. approximately 2.26–2.60 µm in height with a diameter of 3.0– Type strain: descriptions and illustrations serving as type. 4.0 µm. Each sporangium is divided into two unequal sections. GenBank accession number (16S rRNA gene): not reported. The smaller proximal body is not as refractile as the larger, rounded, cup-shaped portion, which encloses an ellipsoidal 5. “Candidatus Pasteuria usgae” Giblin-Davis, Williams, Bekal, endospore broadly elliptic in section having axes of 0.99–1.21 Dickson, Brito, Becker and Preston 2003b, 197 × 1.30–1.54 µm. Endospores seem to be of the kind typical of us’gae. N.L. gen. n. usgae of U.S.G.A., the acronym for the the genus Bacillus; they are resistant to both heat and desicca- United States Golf Association, in gratitude for their financial tion. Nonmotile. Sporangia and vegetative cells are found as support to study this potential biological control agent against parasites in the pseudocoelomic cavities of Meloidogyne spp. The Belonolaimus longicaudatus in turfgrass ecosystems. epithet is now restricted to members of Pasteuria penetrans with Organism is nonmotile with Gram-positive vegetative cells. cup-shaped sporangia and ellipsoidal endospores broadly ellip- Mycelium is septate; hyphal strands branch dichotomously with tic in section occurring primarily as parasites of Meloidogyne spp. expansion of hyphal tip forming sporangium. With scanning Has not been cultivated axenically; the type-descriptive material electron microscopy, peripheral fibers of the mature endospore consists of the text and photographs in Sayre and Starr (1985) protrude around the exposed spherical outer coat of the spore

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creating a crenate border as opposed to other species of Pasteu- ria described from nematodes that have no scalloped border. The sporangium and central body diameters were on average at least 0.5 and 0.7 µm wider than these respective measurements for the other described species of Pasteuria. In lateral view with transmission electron microscopy, the shape of the central body is a rounded-rectangle to a rounded-trapezoid in transverse sec- tion that contrasts with the circular shape for Pasteuria ramosa, the horizontally oriented elliptical shapes for Pasteuria pen- etrans and Pasteuria nishizawae, and the rounded-square shape for Pasteuria thornei. The outer spore coat is thickest laterally, thinner on top and thinnest across the bottom of the spore, being 7–8 times thicker laterally than along the bottom. These measurements contrast with all other described species having outer spore coats with relatively uniform thickness. No basal ring exists around the pore opening as in Pasteuria penetrans. The outer coat wall thickness at its thickest point is >15% (both FIGURE 50. Cauliflower-like, branching, mycelial colony of Pasteuria walls >30%) of the diameter of the central body compared with ramosa attached to the inner walls of the carapace of the cladoceran 3 to <13% (both walls 6 to <25%) for the other described spe- Moina rectirostris. Bar = 10 µm. cies of Pasteuria. The epicortical wall remnant of the mature endospore occurs between the cortex and the inner spore coat in a sublateral band, similar to Pasteuria thornei but different from the other three described species. The epicortical wall in the other described species is as follows: completely concentric in Pasteuria ramosa and Pasteuria nishizawae, and lateral in Pas- teuria penetrans. Obligate endoparasitic bacterium of the pseudocoelom of Belonolaimus longicaudatus that cannot be cultivated on cell-free media. Cultivated only by attachment of endospores to Bacillus longicaudatus and co-cultivation on excised axenic root or green- house plant cultures. Transmission occurs horizontally. Host infection is via cuticular penetration by attached endospores that occurs on all stages of Bacillus longicaudatus except eggs. Sporogenesis, which leads to the death of the host, occurs in the pseudocoelom of J3 through adult stage nematodes. Sporo- genesis is typical of other nematode-specific Pasteuria. Host range appears to be limited to Bacillus longicaudatus, although attachment of endospores has been observed on Bacillus euthy- chilus, but not other soil-inhabiting nematodes. DNA G+C content (mol%): not reported. Type strain: descriptions and illustrations serving as type. GenBank accession number (16S rRNA gene): AF254387. Further information Pasteuria ramosa. When using light microscopy, the earliest visible growth stages of Pasteuria ramosa in the water fleas Daphnia magna and Moina rectirostris Leydig 1860 are the cauliflower-like microcolonies usually found on the inner wall of the inverte-

brate’s carapace Ebert et al., (1996), Sayre et al., 1979; Figure FIGURE 51. Mesosome associated with the septum in a dividing cell of 50). The next detectable stage consists of quartets of sporangia Pasteuria ramosa. Bar = 0.5 µm. carried in the hemolymph throughout the body of the clado- ceran. Later, as the parasite develops, the hemolymph is notice- ably clouded by the myriad immature sporangia of Pasteuria The cell membrane measures 5.8 nm in thickness. Mesosomes ramosa, mainly singles or doublets. (circular membrane complexes) are often found associated Electron micrographs of the “cauliflower” stage reveal circu- with the septa (Sayre et al., 1979; Figure 51). lar patterns of septate hyphal strands of an actinomycete-like The distal or terminal mycelial cells of the microcolonies organism (Figure 49). The hyphae measure approximately enlarge to form teardrop-shaped sporangia that, when viewed 0.67 µm in width and their wall, which is fairly homogeneous by light microscopy, measure approximately 4.8–5.7 × 3.3– in density, is 14.5–15 nm thick. The periplasmic region between 4.1 µm in diameter. Similar materials prepared for transmission the wall and the cell membrane is about 8.7–9.0 nm in width. electron microscopy and sectioned gave measurements of

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3.40–4.35 µm in height and 2.12–2.77 µm in diameter. Early in this process, two septa form; these septa divide the sporangium into anterior, middle, and stem sections (Metchnikoff, 1888; Fig- ure 52). The partitioning is reflected in the outer wall of the sporangia (Figure 53). The anterior section, the upper two-thirds of the sporangium, gives rise to the forespore. Within the anterior section, granu- lar material condenses to form an electron-dense endospore, slightly ellipsoidal to almost spherical in shape, having axes measuring 1.37–1.61 × 1.20–1.46 µm. It comprises a multilay- ered central cytoplasm containing numerous doubled fibrillar strands (Figure 54). Structural changes also occur within the median section, where electron-transparent areas appear to expand and attach laterally to the multilayered endospore wall to form fibrous appendages. The mode of penetration of spores into host cladocerans is not known. Parasitized water fleas (Moina rectirostris) taken from a pond in College Park, MD, USA, were found to yield about 2 × 105 Pasteuria ramosa sporangia per host individual. Generally, the parasite was found in mature females with no young in their egg pouches. Daphnia species, reported previously (Metch- nikoff, 1888) as hosts of this organism, were not parasitized by this bacterial strain (Sayre et al., 1979). Neither sporangia from crushed water fleas nor sediments from the rearing aquaria of

FIGURE 53. Scanning electron micrograph of sporangia of Pasteuria ramosa. External ridges mark boundaries of the endospore, middle sec- tion, and stem of a mature sporangium. Bar = 2.5 µm.

FIGURE 54. Mature sporangium of Pasteuria ramosa containing an endospore, made up of multilayered spore walls (mw), cortex (c), and cytoplast with stranded inclusions (st). Septum (s) separates the stem FIGURE 52. Septa divide the immature sporangium of Pasteuria ramosa from the middle section. The function of the fibrous appendages (wi; into three parts. Bar = 0.5 µm. wing-shaped light areas) is not known. Bar = 0.5 µm.

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parasitized Moina rectirostris (Sayre and Wergin, 1977) resulted in infection when added to healthy populations of Daphnia magna or Daphnia pulex. These two Daphnia species were listed by Metchnikoff (1888) as hosts for Pasteuria ramosa. However, because Moina rectirostris is in the same family (Daphniidae) as these two Daphnia spp., this result may suggest only a very marked host specificity in the particular strain of Pasteuria ramosa available to us, perhaps at the level of a forma specialis, the situation in which one form of a parasite reproduces only in one host species and not in others that are closely related taxo- nomically. Recent work on Pasteuria ramosa from populations of Daphnia magna demonstrated strong specificity (Carius et al., 2001). The type-descriptive material of this taxon (Metchnikoff, 1888) is attached to the form on Daphnia species. If later work should show differences warranting separation at the specific or subspecific level, the taxon on Moina would of course have to be given a different name from Pasteuria ramosa. The influence of water temperature on the occurrence of Pasteuria ramosa in Moina rectirostris in nature was observed over a 3-year period. The parasite was not found until the surface water temperature in the pond reached 26°C or higher, usually about mid-July in the College Park (MD, USA) area. The appar- ent temperature requirement was confirmed in laboratory tests in which water in the aquaria was held at constant temperatures; the parasite was found in 6 and 3 d at 26° and 31°C, respectively, but not at all at 21°C. Pasteuria ramosa from Daphnia magna can be cultured from 15 to 25°C (Ebert et al., 1996). Although endospores of Pasteuria ramosa appear to withstand desiccation, they have only limited resistance to heat. Air-dried sporangia, which were stored for 6 months, were capable of infect- ing healthy populations of cladocerans. However, when air-dried aquarium sediments were heated to 40, 60, or 80°C for 10 min and then added to cultures of healthy cladocerans, they failed to develop after treatment at 80°C (Sayre et al., 1979). Endospores from Pasteuria ramosa from Daphnia populations can survive in pond sediments for decades (Decaestecker et al., 2004). Pasteuria penetrans. Members of nematode-associated Pasteu- ria share several morphological, ultrastructural, and ecologi- cal features Sayre and Starr, 1985; Starr et al., 1983; Starr and Sayre, 1988a; Table 55; Figure 37): all are Gram-positive; all form endospores; all form mycelia, septate and dichotomously branched, vegetative cells; and all parasitize invertebrates (Table 55). The members of the Pasteuria penetrans group differ in many respects from Pasteuria ramosa: colony shape, shape and size of sporangia (Figure 55) and endospores, and host relations FIGURE 55. Drawings of sections through typical mature sporangia of (Table 56). Upon recognition (Sayre and Starr, 1985) of its rela- three Pasteuria species emphasizing ultrastructural features useful in identification. A, Pasteuria ramosa, parasite of cladoceran water fleas. tionship to the genus Pasteuria, the first of these nematode para- B, Pasteuria penetrans sensu stricto, parasite of Meloidogyne incognita. C, sites to receive such taxonomic attention was renamed Pasteuria Pasteuria thornei, parasite of Pratylenchus brachyurus. Labels: protoplast penetrans (ex Thorne) Sayre and Starr (1985). Subsequently, the (p) containing stranded inclusions; multilayered structures including a name Pasteuria penetrans sensu stricto (i.e., in the strict sense) was cortex (c) and inner (isc) and outer (osc) spore coats; parasporal fibers limited in scope to the bacterium parasitic on Meloidogyne spp. (pf); granular matrix (m); sporangial wall (sw); in Pasteuria ramosa, a and particularly Meloidogyne incognita (Starr and Sayre, 1988a). stem cell (sc) remains attached to the sporangium. A second species, Pasteuria thornei, was erected for parasites of the lesion nematodes of the genus Pratylenchus and particularly Pasteuria brachyurus (Starr and Sayre, 1988a). Figure 58); where substantial differences have been observed, Much of the following information stems from studies of they are noted below. Cross-sections viewed by transmission elec- Pasteuria penetrans. Pasteuria thornei, which has received much tron microscopy (Imbriani and Mankau, 1977; Sayre and Starr, less study than its relative, is similar in most respects examined 1985; Sayre and Wergin, 1977; Starr and Sayre, 1988a) reveal (transmission electron micrographs of sporangia of Pasteuria that the endospore of Pasteuria penetrans sensu stricto consists of a thornei at various stages are shown in Figure 56, Figure 57, and central, highly electron-opaque core surrounded by an inner

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FIGURE 56. Transmission electron micrograph of Pasteuria thornei parasitizing a juvenile of the lesion nematode Pratylenchus brachyurus, showing simultaneous occurrence of vegetative microcolonies (MC) and sporangia (SP) of the bacterium in the nematode’s pseudocoelo- mic cavity. Bar = 1.0 µm.

FIGURE 58. Lateral view of a mature sporangium of Pasteuria thornei showing inner sublateral cortical wall (arrows) that gives the endospore an angular appearance. The basal cortical wall for the endospore thins to provide a germinal pore. The basal portion of the sporangium con- tains an irregular granular matrix intermingled with fibrillar strands. Bar = 0.5 µm.

from the sides of the endospore to the cuticle of the nematode, where they become more electron-dense. A mature endospore of Pasteuria penetrans sensu stricto attaches to the surface of a nematode so that a basal ring of wall mate- rial lies flatly against the cuticle. A median section through the endospore and perpendicular to the surface of the nematode bisects this basal ring. As a result, the ring appears as two pro- truding pegs, which are continuous with the outer layer of the spore wall and rest on the cuticular surface of the nematode (Figure 43). The peripheral fibers of the endospore also are closely associated with the nematode’s cuticle. These fibers, which encircle the endospore, lie along the surface of the nematode and follow the irregularities of the cuticular annuli. They seem not to penetrate the cuticle. The germ tube of the endospore of Pasteuria penetrans emerges through the central opening of FIGURE 57. Section of immature sporangium of Pasteuria thornei the basal ring, penetrates the cuticle of the nematode, and showing the developing septum (SE) separating the polar area that enters the hypodermal tissue (Figure 43). Hyphae were initially condenses into an endospore from the parasporal matrix; the light encountered beneath the cuticle of the nematode near the areas sublateral to the polar area will develop into parasporal fibers. Bar = 0.5 µm. site of germ-tube penetration. From this site, they apparently penetrate the hypodermal and muscle tissues and enter the pseudocoelom. and an outer wall composed of several distinct layers (Figure 48). Mycelial colonies of Pasteuria penetrans up to 10 µm in diam- When observed with the transmission electron microscope, the eter are formed in the pseudocoelom, where they are observed peripheral matrix of the spore is fibrillar. Fine microfibrillar after the diseased juvenile penetrates plant roots (Figure 44). strands, about 1.5 nm thick, extend outward and downward The hyphae comprising the colony are septate. A hyphal cell,

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which is 0.40–0.50 µm in cross-section, is bounded by a compound Other organisms – “Pasteuria hartismerei”. Pasteuria hartismerei wall, 0.12 µm thick, composed of an outer and an inner mem- Bishop, Gowen, Pembroke and Trotter (2007) was described brane. The inner membrane of the wall forms the septation from Meloidogyne ardenensis on the basis that the endospores and delineates individual cells. In addition, this membrane is lacked a basal ring on their ventral side and they had a unique continuous with a membrane complex or mesosome that is clumping nature inside their host (Bishop et al., 2007). Also, frequently associated with the septum (Figure 45). Because of a unique 16S rRNA sequence was obtained which differs from the sinuous and branching growth habit, cell length cannot be that of other described Pasteuria. This taxon will become a Can- determined from thin section electron micrographs. didatus species when validated according to the International Sporulation of Pasteuria penetrans occurs in a developmental Code of Nomenclature of Bacteria (1992) (Euzéby, 1998). pattern that is similar to that observed in axenic cultures of Bacil- References lus spp., e.g., Bacillus subtilis. During the controlled and semi- synchronous growth of Meloidogyne arenaria race 1 infected with a Anderson, J.M., J.F. Preston, D.W. Dickson, T.E. Hewlett, N.H. Wil- line (P20) of Pasteuria penetrans, endospore maturation was coin- liams and J.E. Maruniak. 1999. Phylogenetic analysis of Pasteuria pen- cident with the formation of spore-associated adhesin, as deter- etrans by 16S rRNA gene cloning and sequencing. J. Nematol. 31: mined by a specific monoclonal antibody (Brito et al., 2003). 319–325. Atibalentja, N., G.R. Noel and L.L. Domier. 2000. Phylogenetic position Sporulation of Pasteuria penetrans is initiated in the female of the North American isolate of Pasteuria that parasitizes the soy- nematode. As the process begins, the terminal hyphal cells of bean cyst nematode, Heterodera glycines, as inferred from 16S rDNA the mycelium bifurcate and enlarge from typical hyphal cells sequence analysis. Int. J. Syst. Evol. Microbiol. 50: 605–613. to ovate cells measuring 2.0 × 4.0 µm. Structure and content of Atibalentja, N., B.P. Jakstys and G.R. Noel. 2004a. Life cycle, ultrastruc- the cytoplast change from a granular matrix, which contains ture, and host specificity of the North American isolate of Pasteu- numerous ribosomes as found in the hyphal cells, to one lack- ria that parasitizes the soybean cyst nematode, Heterodera glycines. J. ing particulate organelles. During these changes, the develop- Nematol. 36: 171–180. ing sporangia separate from their parental hyphae, which cease Atibalentja, N., G.R. Noel and A. Ciancio. 2004b. A simple method for to grow and eventually degenerate. PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA After these early structural alterations, a membrane forms from small numbers of endospores. J. Nematol. 36: 100–105. Atibalentja, N. and G.R. Noel. 2008. Bacterial endosymbionts of plant- within the sporangium and separates the upper third of the cell parasitic nematodes. Symbiosis (in press). or forespore from its lower or parasporal portion (Figure 46). Baermann, G. 1917. Eine einfache Methode zur Affindung von Anky- The granular matrix confined within the membrane then con- lostomun (Nematoden) Larven in Erdproben. Geneesk. Tijdschr. denses into an electron-opaque body, 0.6 µm in diameter, which Nederl. Indiue 57: 131–137. eventually becomes encircled by a multilayered wall. The result- Bekal, S., R.M. Giblin-Davis and J.O. Becker. 1999. Gnotobiotic culture ing discrete structure is an endospore. of Pasteuria sp. on Belonolaimus longicaudatus. J. Nematol. 31: 522. Coincident with the formation of an endospore in Pasteuria Bekal, S., J. Borneman, M.S. Springer, R.M. Giblin-Davis and J.O. penetrans is the emergence of parasporal fibers. These fine fibers, Becker. 2001. Phenotypic and molecular analysis of a Pasteuria strain which form around the base of the spore, differentiate from an parasitic to the sting nematode. J. Nematol. 33: 110–115. electron-translucent, granular substance. They appear to con- Bird, D.M., C.H. Opperman and K.G. Davies. 2003. Interactions between bacteria and plant-parasitic nematodes: now and then. Int J nect with and radiate from the external layer of the wall of the Parasitol 33: 1269–1276. endospore (Figure 47). During development of the parasporal Bishop, A.H. and D.J. Ellar. 1991. Attempts to culture Pasteuria penetrans fibers, the formation of another membrane, the exosporium, in vitro. Biocontrol Sci. Technol. 1: 101–114. isolates the newly formed endospore within the sporangium. At Bishop, A.H., S.R. Gowen, B. Pembroke and J.R. Trotter. 2007. Mor- this later stage of spore development, the granular content of phological and molecular characteristics of a new species of Pasteuria the paraspore becomes less dense, degenerates, and disappears. parasitic on Meloidogyne ardenensis. J. Invertebr. Pathol. 96: 28–33. As a result, the mature sporangium contains a fully developed Brito, J.A., J.F. Preston, D.W. Dickson, R.M. Giblin-Davis, D.S. Williams, endospore enclosed within the exosporium (Figure 48). H.C. Aldrich and J.D. Rice. 2003. Temporal formation and immu- The cell wall of the sporangium of Pasteuria penetrans remains nolocalization of an endospore surface epitope during Pasteuria pen- intact until the remnants of the infected nematode are dis- etrans sporogenesis. J. Nematol. 35: 278–288. Brito, J.A., T.O. Powers, P.G. Mullin, R.N. Inserra and D.W. Dickson. rupted, after which event the endospores are released. The 2004. Morphological and molecular characterization of Meloidogyne exosporium apparently remains associated with the endospore mayaguensis isolates from Florida. J. Nematol. 36: 232–240. until contact is made with a new nematode and the infection Buchanan, R.E. 1925. General Systematic Bacteriology. The Williams cycle restarts. The vermiform juvenile stages of the nematodes and Wilkins Co., Baltimore. are encumbered by the parasite as they migrate through soils Butler, M.K., J. Wang, R.I. Webb and J.A. Fuerst. 2002. Molecular and infested by the endospores of Pasteuria penetrans sensu stricto and ultrastructural confirmation of classification of ATCC 35122 as a the infectious cycle is repeated. strain of Pirellula staleyi. Int. J. Syst. Evol. Microbiol. 52: 1663–1667. Much morphological, ultrastructural, developmental, and Canning, E.U. 1973. Protozoal parasites as agents for biological control host diversity is evident in Pasteuria (Giblin-Davis et al., 2001; of plant-parasitic nematodes. Nematologica 19: 342–348. Sayre and Starr, 1985; Sayre et al., 1988). We believe this diversity Carius, H.J., T.J. Little and D. Ebert. 2001. Genetic variation in a host- parasite association: potential for coevolution and frequency-depen- speaks for the existence of many taxa within the group. Pasteu- dent selection. Evolution Int. J. Org. Evol. 55: 1136–1145. ria thornei, Pasteuria nishizawae, and “Candidatus Pasteuria usgae” Charles, L., I. Carbone, K.G. Davies, D. Bird, M. Burke, B.R. Kerry and represent three cases where novel species have been proposed C.H. Opperman. 2005. Phylogenetic analysis of Pasteuria penetrans by on substantial morphological, morphometric, host range, and use of multiple genetic loci. J Bacteriol 187: 5700–5708. molecular grounds (Table 56) (Giblin-Davis et al., 2001; Giblin- Chen, S.Y., J. Charnecki, J.F. Preston and D.W. Dickson. 2000. Extraction Davis et al., 2003b; Sayre et al., 1988; Starr and Sayre, 1988a). and purification of Pasteuria spp. endospores. J. Nematol. 32: 78–84.

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