Spiroplasma Poulsonii Sp. Nov., a New Species Associated with Male-Lethality in Drosophila Willistoni, a Neotropical Species of Fruit Fly

International Journal of Systematic Bacteriology (1999), 49, 61 1-6 18 Printed in Great Britain

Spiroplasma poulsonii sp. nov., a new species associated with male-lethality in Drosophila willistoni, a neotropical species of fruit fly

David L. Williamson,'t Bungo Sakaguchi,2 Kevin J. Ha~kett,~ Robert F. Whit~omb,~$Joseph G. T~lly,~Patricia Carle,6 Joseph M. Bo~e,~ Jean R. ad am^,^ Meghnad Konai3§and Roberta B. Henegar3

Author for correspondence: David L. Williamson. Tel: + 1 516 724 0897. Fax: + 1 516 444 3947. e-mail: [email protected]

Department of Progenies from some wild-caught females of Drosophila willistoni and three Ana tom i ca I Sciences, other sibling species are entirely female. The proclivity for production of State University of New York, Stony Brook, unisexual female progeny by these flies was named the sex ratio (SR) trait and NY 11794, USA was originally thought to be genetic. However, experiments in the laboratory * Faculty of Agriculture, of Donald F. Poulson in the early 1960s demonstrated that this 'trait' was Kyushu University, vertically transmitted and infectious, in that it could be artificially transferred Fukuoka, Japan by injection from infected females to non-infected females. Motile, helical 3,4 Insect Biocontrol micro-organisms were observed in females showing the trait. In 1979, the SR La boratory3 and organisms were designated as group II in the informal spiroplasma Vegetable Laboratory4, US Department of classification system. The organisms proved to be extremely fastidious, but Agriculture, Beltsville, were eventually cultivated in a very complex cell-free medium (H-2) after MD 20705, USA initial co-cultivation with insect cells. Cultivation in the H-2 medium and the Mycoplasma Section, subsequent availability of a triply cloned strain (DW-19 permitted comparative Laboratory of Molecular studies. Cells of strain DW-lTwere helical, motile filaments 200-250 nm in Microbiology, National Institute of Allergy and diameter and were bound by a single trilaminar membrane. Cells plated on Infectious Diseases, 1.8% Noble agar formed small satellite-free colonies 60-70 pm in diameter Frederick Cancer Research with dense centres and uneven edges. The temperature range for growth was FaciI ity, Frederick, MD 21702, USA 26-30 "C; optimum growth occurred at 30 "C, with a doubling time in H-2 medium of 15.8 h. The strain passed through filters with 220 nm, but not 6 Laboratoire de Biologie Cellulaire et Mol&ulaire, 100 nm, pores. Reciprocal serological comparisons of strain DW-lTwith lnstitut National de la representatives of other spiroplasma groups showed an extensive pattern of Recherche Agronomique one-way crossing when strain DW-lTwas used as antigen. However, variable, and Universite de Bordeaux II, 33883 usually low-level reciprocal cross-reactions were observed between strain DW- Villenave d'Ornon Cedex, lTand representatives of group I sub-groups. The genome size of strain DW-lT France was 2040 kbp, as determined by PFGE. The G+C content was 2621 mol%, as determined by buoyant density and melting point methods. The serological and molecular data indicate that strain DW-lTis separated from group I representative strains sufficiently to justify retention of its group status. Continued group designation is also indicated by the ability of SR spiroplasmas to induce male lethality in Drosophila, their vertical transmissibility and their extremely fastidious growth requirements. Group II spiroplasmas, represented by strain DW-lT(ATCC 431533, are designated Spiroplasma poulsonii.

Keywords : Spiroplasma poulsonii sp. nov., mollicute, sex ratio, Drosophila willistoni

., . , ., ...... , . , . . , ...... ,. , . . . , . . , ., . , . . . . , ., .. . . , , .. . , ...... , ...... , . . . , .. . . , .. . , , .. . . , . . . . , . . . , . . , . , . . , . , . . . . . , ...... , ...... , , ...... , ...... , ...... , .. , . , , ...... , ...... I.. . ?Present address: 4 Galahad Lane, Nesconset, NY 11767-2220, USA. $Present address: Carl Hayden Bee Research Center, USDAIARS, 2000 E Allen Road, Tucson, A2 85719, USA. $Present address: 9125 Tymat Court, Laurel, MD 20723, USA. Abbreviation: SR, sex ratio.

00943 0 1999 IUMS 61 1 D. L. Williamson and others

INTRODUCTION Prior to the establishment of a cloned isolate of the D. willistoni SR spiroplasma, studies on the organisms A curious relationship between spiroplasmas and were performed using suspensions of spiroplasmas in insects occurs in natural populations of four closely Drosophila. physiological saline, which were prepared from small related neotropical species of In collections samples of haemolymph aspirated from D. willistoni of flies of the Drosophila willistoni group from tropical flies carrying the SR trait. Microscopic enumeration of South America and islands of the Antilles, single haemolymph dilutions indicated that haemolymph females were found whose progenies consisted of only titres in D. willistoni were about 1 x 10l1 p1-l. Sus- daughters (Poulson, 1963). This trait, the sex ratio pensions of organisms from fly haemolymph were used (SR) trait, is widely distributed among members of the to produce an antiserum in rabbits, and to determine D. willistoni : D. willistoni species group from Jamaica the G+C content of DNA from the uncloned ;Drosophila nebulosa ; and Brazil from Haiti and Brazil organisms. Vigorous attempts were made to cultivate Drosophila equinoxialis subsp. equinoxialis from Brazil caribbensis the SR spiroplasma, but even though certain medium and subsp. from Puerto Rico, the formulations permitted them to be maintained for Dominican Republic and Jamaica ; and Drosophila paulistorurn varying periods, none provided the necessary factors from Colombia, Brazil and the West for growth (Williamson et al., 1983). Finally, however, Indies. Early studies of the SR trait conducted in the in 1986, Hackett and collaborators reported that they laboratory of Professor Donald F. Poulson at Yale had successfully grown and cloned the D. willistoni SR University demonstrated that the SR trait in the D. et al., willistoni spiroplasma (Hackett 1986). In these studies, the species group was maternally (vertically) SR spiroplasma was first grown in co-culture with a inherited and infectious, in that it could be artificially lepidopteran cell line and subsequently in cell-free transferred by injection from infected females to non- culture in a new medium, H-2 (Hackett et al., 1986). infected females, who then transmitted the trait to & ; Cultivation of the organisms permitted them to be their daughters (Malogolowkin Poulson, 1957 cloned, and the DW-lT strain was selected for further Malogolowkin et al., 1959; Sakaguchi & Poulson, SR Drosophila study. After injection of a suspension of strain DW-IT 1963). An extensive review of the trait in spiroplasmas into Drosophila pseudoobscura Pifion and the organisms that induce it was published in 1979 & Normal strain females, their progenies became entirely (Williamson Poulson, 1979). female (Williamson et al., 1989). However, the cloned In 1960, one of us (B. Sakaguchi), during a post- organisms had lost the ability to be vertically trans- doctoral fellowship in Professor Poulson’s laboratory, mitted. discovered the infectious nature of the SR trait. Using a glass micropipette, a small sample of haemolymph Reciprocal spiroplasma deformation and metabolism was aspirated from D. willistoni females producing inhibition tests involving strain DW- 1 and represen- unisexual (female) progenies and placed under a drop tative strains of all other groups and sub-groups have of crown immersion oil on a microscope slide and now been completed. These tests showed strong observed, first under ordinary light microscopy, by patterns of one-way cross-reactions when strain DW- which nothing was visible, and subsequently under lTwas used as antigen (Williamson et al., 1998). Some phase-contrast microscopy. Even though the optics of reciprocal crossing patterns between strain DW- 1 this microscope provided much less contrast than the and group I spiroplasmas were observed, but the optics of the dark-field microscopes currently used for patterns were not as strong as most intragroup spiroplasma observation, Sakaguchi was surprised to reactions among group I spiroplasmas. The phylo- find that the haemolymph was teeming with small genetic relationship of strain DW-lT to other spiro- filamentous organisms. In freshly mounted pre- plasmas was clarified by the studies of Weisburg et al. parations, waves of helicoidal movement could be seen (1989). These studies showed that group I and group I1 to pass along the length of the filaments, giving them a spiroplasmas were more closely related to each other regular spiral appearance. Fixation of such pre- than to other spiroplasma groups, but nonetheless had parations over formaldehyde vapour, followed by significant sequence dissimilarity. The status of strain various staining procedures, suggested that the DW-IT as the representative strain of group I1 spiro- organisms were similar in size and general aspect to plasmas is therefore justified on the basis of molecular spirochaetes in the genus Treponerna (Poulson & and serological data. In addition, its unique biological Sakaguchi, 1961). Hence, the organisms were referred characteristics - host specificity, pathogenicity for to as ‘sex ratio spirochaetes’, until they were shown male progeny, transovarial transmission and fastidious (Williamson, 1969 ;Williamson & Whitcomb, 1974) by growth requirements - also augur for its group status. electron microscopic examination of fixed and nega- In this report we present the results of characterization tively stained cells to be devoid of axial filaments and studies of strain DW-lT in fulfillment of the minimal outer envelopes - structures characteristic of spiro- standards for new species descriptions for strains chaetes. Instead, the SR organisms proved to be classified in the class Mollicutes (International Com- structurally similar to spiroplasmas associated with mittee on Systematic Bacteriology Subcommittee on corn stunt disease (Davis et al., 1972; Chen & Liao, the taxonomy of Mollicutes, 1995). Strain DW-lT is 1975; Williamson & Whitcomb, 1975) and stubborn hereby designated as the type strain (ATCC 43 153T) disease of citrus (Cole et al., 1973). of a new species, Spiroplasrna poulsonii, named in

61 2 International Journal of Systematic Bacteriology 49 Spiroplasma poulsonii sp. nov.

strains of all currently recognized groups and sub-groups, including the type strains of named species (Williamson et al., 1998), were utilized for these comparative studies. Culture medium and cultivation techniques. H-2 medium (Hackett et al., 1986), a combination of I part DCCM (Hackett & Lynn, 1995), 2 parts MID (Whitcomb, 1983) and 1 part modified Grace's Insect Medium (TNM-FH) (Hackett & Lynn, 1985), supplemented with 15% (v/v) foetal bovine serum and containing 800 U penicillin ml-', was used as the growth medium for all tests involving strain DW- I '. Batch cultures of cells in this medium were prepared in 9 ml H-2 medium. About 10 d were required to reach maximum titre (about 1 x lo7ml-', initially). To prepare solid H-2 medium, 8 YO(w/v) Noble agar (Difco) was added to the autoclavable fraction of MlD, microwaved to melt the agar, equilibrated at 56°C and added to H-2 broth medium at 56 "C to produce a final agar concentration of 1.8%. Cultures of strain DW-IT on solid media were incubated at 26 "C, either aerobically without added CO, or with 5% (v/v) CO, (Enhanced CO, Gaspak system, BBL Microbiology Systems) or anaerobically (Hydrogen and CO, Gaspak system). The SP-4 and MID medium formulations were used for the cultivation of other spiroplasma Fig. 1. Colony of strain DW-lT on H-2 medium supplemented strains. Temperature requirements for growth were de- with 1.8% Noble agar after 32 d at 26 "C under aerobic termined by the method of Konai et al. (1996). conditions with 5 % CO,. Bar, 20 pm. Morphological studies. The motility and gross morphology of strain DW-lTcells were determined by observing them in 3-5 p1 samples of broth cultures under dark-field illumi- nation at x 800-1250. For electron microscopic studies of memory of Professor Donald F. Poulson, in whose the fine structure of strain DW- lTcells, previously published laboratory at Yale University SR organisms were procedures (Williamson, 1983) were followed. first studied. Sterol requirements. The sterol requirement for growth was evaluated by determining the protein yield of strain DW-IT grown in serum-free H-2 broth supplemented with increasing METHODS amounts of foetal bovine serum following procedures previously described (Tully, 1983b). Spiroplasma strains. Strain DW- 1 was cultivated (Hackett et al., 1986) from a haemolymph suspension obtained from Tests for biological and biochemical properties. The ex- D.pseudoobscura (Piiion Normal strain) female flies arti- tremely fastidious nature of strain DW- lT precluded ficially infected by injection of naturally infected Barbados- adaptation in base broth media usually employed for the 3 strain D.willistoni SR haemolymph. Briefly, 2-5 pl hae- demonstration of glucose fermentation and hydrolysis of molymph from the infected females was collected by glass arginine and urea (Aluotto et al., 1970). The filtration needle and transferred to 500 p1 M1D medium. In D. characteristics were measured in H-2 broth by procedures pseudoobscura, SR organisms reach a titre of about previously reported (Tully, 1983a). x 1 loll pl-', so the initial cell titre in the primary culture was Serological tests. Cell pellets of organisms were prepared by about 5 x lolo p1-l. An aliquot of this suspension was mixed growing bulk cultures in 500 ml H-2 media. Homologous with lepidopteran cell culture medium (DCCM) (Hackett & antiserum against strain DW-lTwas produced in rabbits as Lynn, 1995) and further aliquoted to microtitre wells previously described (Williamson et al., 1979). Hyper- containing embryo-derived cells from the cabbage looper immune antisera against all established Spiroplasrna species, (Trichoplusia ni) cell line (IPLB-TN-R2) (Rochford et al., groups and sub-groups (Williamson et al., 1998) were 1984). Primary isolation was accomplished in co-culture obtained from reference collections at the USDA Beltsville with T. ni cells initially growing in TNM-FH medium Agricultural Research Center, Beltsville, MD, USA and the (Hackett et al., 1986). Organisms growing in these cultures National Institute of Allergy and Infectious Diseases lab- were eventually adapted to grow in the cell-free H-2 medium oratory, Frederick, MD, USA. These antisera and their (Hackett et al., 1986). Growth was evaluated by enumeration respective antigens were tested against strain DW-IT in of helical cells in 3 or 5 pl samples under dark-field reciprocal deformation and metabolism inhibition tests microscopy (Williamson, 1969). After nine co-culture (Williamson et al., 1978, 1979). passages, the SR organisms were adapted to grow in cell-free H-2 medium. At passage 20, the spiroplasmas were triply Genome analysis. The techniques for extraction and puri- cloned by the limiting dilution method of Whitcomb & fication of chromosomal DNA from strain DW-lT have Hackett (1986). One triply cloned strain at passage 23 was been described previously (Carle et al., 1983a). The DNA selected and designated strain DW-1' (ATCC 43153T). A G+C composition of strain DW-IT was determined by batch culture was prepared, aliquoted to ampules and buoyant density and melting temperature following the lyophilized. Rehydrated cultures were used for the charac- procedures of Carle et al. (1983b). G+C determinations terization studies reported here. Representatives of the were performed first on pelleted uncloned organisms and

International Journal of Systematic Bacteriology 49 613 D. L. Williamson and others later on pelleted organisms of strain DW-IT. The genome size of strain DW-ITwas determined by PFGE, employing techniques described earlier (Carle et al., 1995). Purified DNA from Spiroplasma citri (genome size 1820 kbp ; G + C content 26f 1 mol%) was used as a reference in all procedures (Carle et al., 1995).

RESULTS AND DISCUSSION Cu Itu ral and morphological properties Strain DW-lTgrew to titres of lo7-lo8 organisms ml-I in H-2 medium. All attempts to adapt strain DW-ITin other media commonly employed for spiroplasma cultivation (Whitcomb, 1983) were unsuccessful : organisms failed to grow beyond the first 10-fold dilution when inoculated into MID or SP-4 media or into basal broth (BSR) medium containing either 10 YO foetal bovine serum or 1 YO (v/v) bovine serum fraction. The doubling time for strain DW-lT growing in H-2 medium was 15-8h at 30 "C, its optimum temperature for growth. This is one of the longest doubling times for spiroplasmas in culture. Strain fig. 2. Electron micrograph of a sectioned and stained pellet of DW-IT grew on solid medium on 1-8% Noble agar strain DW-lT cells. The section was stained with 2% aqueous plates incubated aerobically with 5% CO, at 26 "C. uranyl acetate and Reynold's lead citrate. The arrow indicates Colonies were first evident 32 d after inoculation; fully the unit membrane. Bar, 200 nm. developed colonies were small (60-70 pm in diameter) with dense centres and uneven edges (Fig. 1); no satellite colonies were observed. cultures of strains that utilize arginine, has not been Dark-field microscopy of broth cultures of strain DW- observed. Filtration of strain DW-IT broth cultures IT showed motile helical filaments with four or more with a cell titre of 1 x lo7 colour-changing units turns. Electron microscopy of thin sections of pellets of (CCUs) ml-l through 450, 300 and 220 nm pore-size uncloned organisms from insect haemolymph membrane filters resulted in cell titres of lo7, lo6 and & (Williamson Whitcomb, 1974) and of strain DW-IT lo6CCUs rnl-', respectively. There were no viable cells (Fig. 2) revealed the presence of filamentous cells in the 100 nm pore-size membrane filtrate. The end 200-250 nm in diameter bounded by a single trilaminar point of the unfiltered dilution series was reached after membrane. 70 d culture.

Sterol requirement Serological tests The amounts of protein contained in pellets of strain The results of reciprocal spiroplasma deformation DW- lT inoculated into H-2 medium supplemented YO (DF) and metabolism inhibition (MI) tests of strain with 15, 10 and 2 foetal bovine serum were 0.52,0.54 DW- 1 versus representatives of the currently recog- and 0.22 mg per 100 ml, respectively. No protein nized strains of the groups and sub-groups of spiro- production or colour change (see below) was observed plasmas were published in a recent revision of the in media containing 0% foetal bovine serum, and no group classification of the genus Spiroplasma spiroplasma cells were found in 50 fields examined et al., (5 (Williamson 1998). Serological data pertaining under dark-field microscopy pl culture sample). to strain DW-IT are presented in Table 1. In the DF These results indicate that serum or sterol is required test, when strain DW-IT was used as an antigen, titres for growth of strain DW-IT. of 20-640 were obtained with the antisera to group I representatives ;four sera to other spiroplasma groups Biochemical and biological properties cross-reacted at low levels. Serum directed against strain DW-lT reacted with antigens from six of the Growth of strain DW-IT in H-2 medium, which group I representatives, with a maximum titre of 80, contains 0-025YO (v/v) phenol red as a pH indicator, is and with PUP-IT (group XIX). The titre in the accompanied by a colour shift from red-orange to homologous reaction was 1280. In the MI test, when yellow as the number of cells increases, indicating that strain DW-lTwas used as antigen, titres of 162-39000 acid is produced from one or more of the various were observed against group I antisera. However, substrates contained in the medium. A subsequent some reactivity was observed with almost all spiro- shift back to red-orange colour, as is observed in plasma antisera. Outside group I, a titre of 1458 was

614 international Journal of Systematic Bacteriology 49 Spiroplasrna poulsonii sp. nov.

Table 1. Serological reactivities of the group II spiroplasma strain DW-lT

Antigen and antisera of the strains were tested in all heterologous combinations against all known and putative groups and sub- groups. All cross-reactions were negative (-) except where shown. Values in the ‘Antigen’ columns are reciprocals of the end points when antiserum to DW-ITwas used against the designated antigen. Values in ‘Antiserum’ columns are reciprocals of end points when DW- 1 was used as antigen against heterologous antiserum.

Group Strain Deformation Metabolism inhibition

Antigen Antiserum Antigen Antiserum

I- 1 Maroc R8A2T 162 1-2 BC-3T 486 1-3 E275T 486 1-4 277F 4 374 1-5 LB- 12 39 000 1-6 M55T 4 374 1-7 N525 39 000 1-8 P40T 1458 11 DW-lT > 117000* 111 23-6T 18 IV B31T 54 VI Y32T 18 VII MQ- 1 54 VIII- 1 EA- 1 54 VIII-2 DF- 1 1458 IX CN-ST 162 X AES- 1 486 XI1 DU- 1 18 XI11 Ar- 1343T 54 xv 1-25 54 XVI- 1 CC-IT - XVI-2 CB- I 54 XVI-3 Ar-1357 54 XVIII TN- 1 18 XIX PUP- 1 18 xx LD- 1 54 XXI W115 18 XXII CT- 1 18 XXIII TG- 1 54 XXIV CCHT 18 xxv CUAS- 1 18 XXVI PLHS- 1 - XXIX TIUS- 1 18 xxx BIUS- 1 18 XXXI HYOS-lT 54 XXXII TABS-2T 18 XXXIII TAUS- 1 18 XXXIV BARC 1901 486 Ungrouped BARC 2649 1458 *Homologous titres of strain DW-IT in the respective test system. The reaction, obtained only once, is listed in each column for completeness.

obtained when strain DW-lT reacted with serum 1-8) was inhibited by antiserum directed against strain against strain DF-lT (sub-group VIII-2; Spiroplasrna DW-IT. The homologous titre in the MI test was chrysopicola) and with serum against ungrouped strain > 1 17 000. When the DF and MI tests are considered BARC 2649. When strain DW-IT was represented as together, only one set of reactions, those with sub- antiserum, growth of only two group I strains (1-2 and group 1-8 (Spiroplasrna phoeniceurn) showed complete

International Journal of Systematic Bacteriology 49 61 5 D. L. Williamson and others reciprocal cross-reactivity, and then only at low titre. 1986) ; sub-group 1-6 (Spiroplasma insolitum ; Hackett This profile of serological cross-reactivity is consider- et al., 1993); and sub-group 1-8 (S. phoeniceum; ably weaker than that observed among the sub-groups Saillard et al., 1987). Therefore, minor serological of group I (Whitcomb et al., 1982), suggesting a more cross-reactivity is not a barrier to separate species distant link between group I sub-groups and strain designation, even for spiroplasma sub-groups. In the DW-lT than among the group I sub-groups. case of the group I sub-groups, species designations were supported by DNA-DNA homology data. In the case of the group I1 strain DW-lT,separation from the Genome size and DNA base composition group I spiroplasmas is indicated by thel6S rRNA The base composition (G + C content) of strain DW- gene sequencing data (GenBank M24483) of Weisburg lT was 26f 1 mol%. The same value was obtained et al. (1989), which showed substantial sequence from analysis of DNA from uncloned organisms. The dissimilarity between group I and group I1 strains. undigested linear DNA of strain DW-lTwas 2040 kbp, This divergence has recently been recalculated (Hurst as determined by PFGE. et al., 1999) and found to be about 2-5 YO.Not only the molecular data, but biological data, including the extremely fastidious growth requirements, host re- Habitat and pathogenicity striction, male lethality and vertical transmissibility of Haemolymph is the normal habitat of the D.willistoni uncloned organisms, indicate that the group I1 des- SR spiroplasma, where it grows to titres of approxi- ignation of strain DW- 1 should be preserved to reflect mately loll organisms p1-l. The ability of strain DW- its unique status. As a result of the characterization studies described herein, the name Spiroplasma lTto grow in haemolymph, to eliminate male progeny poulsonii from injected females and to be vertically transmitted is proposed for the group I1 representative, to the subsequent generation was tested (Williamson et strain DW- 1'. The taxonomic description below al., 1989) by injection of D. pseudoobscura females with summarizes the properties of this new species. a suspension of strain DW-lT spiroplasmas at the sixteenth passage after triple cloning (39 total Description of Spiroplasma poulsonii sp. nov. passages). In these studies, strain DW-IT was capable Spiroplasrna poulsonii (pou1.son'i.i. M.L. n. poulsonii of growing in the haemolymph and causing the of Poulson, named in memory of Donald F. Poulson, elimination of male progeny, but had lost the ability to be vertically transmitted. Professor of Biology at Yale University, in whose laboratory this spiroplasma was discovered and stud- Properties described herein for strain DW- lTfulfil the ied intensively). proposed criteria (International Committee on Sys- Cells are motile helical filaments 200-250 nm in di- tematic Bacteriology Subcommittee on the taxonomy ameter. The membrane-bound cells lack cell walls. Mollicutes, Mollicutes, of 1995) for species of the class Cells plated on 1-8% Noble agar form small satellite- including the absence of a cell wall, filterability, the free colonies 60-70 pm in diameter that have dense lack of reversion to walled bacteria when grown in centres and uneven edges. Chemo-organotroph. Acid antibiotic-free media, and penicillin resistance. The is produced during growth in H-2 medium. The helicity and motility of strain DW-lT place it in the temperature range for growth is 2630 "C, with op- order Entomoplasmatales and the family Spiro- timum growth at 30 "C. The doubling time at the plasmataceae (Tully et al., 1993). The results of the optimum temperature in H-2 medium is 154 h. Sero- serological comparison of strain DW-lT with other logically distinct from other established Spiroplasma Spiroplasma species and groups indicate that strain species. Isolated from the haemolymph of Drosophila DW-lTshares antigens with group I sub-group strains. pseudoobscura females infected by haemolymph trans- Most of the cross-reactions involving these antigens fer of the Barbados-3 strain of Drosophila willistoni SR were much stronger when strain DW-IT served as organism. Pathogenicity (lethality to male progeny) antigen. Strain DW-IT, when used as antigen, also confirmed by injection into Drosophila pseudoobscura. cross-reacted with antisera directed against repre- Vertical transmissibility lost after cultivation and sentatives of many other spiroplasma groups. The cloning. The G+C content of the DNA is property of extensive cross-reaction with many anti- 26+ 1 mol%, as determined by buoyant density and spiroplasma sera has also been observed with Spiro- melting point methods. The genome size is 2040 kbp, plasma lampyridicola (Stevens et al., 1997), Spiro- plasma alleghenense et al., as determined by PFGE. The GenBank accession (Adams 1997) and, to a number for the 16s rRNA gene sequence is M24483. somewhat lesser degree, Spiroplasma platyhelix The type strain is DW-IT (=ATCC 43153T). (Williamson et al., 1997). Levels of cross-reaction greater than the intergroup reactions between groups I and I1 occur among the sub-groups of group I, but REFERENCES have not prevented the designation of separate species Adams, J. R., Whitcomb, R. F., Tully, 1. G. & 7 other authors for sub-groups 1-1 (S. citri; Saglio et al., 1973); sub- (1997). 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61 6 International Journal of Systematic Bacteriology 49 Spiroplasma poulsonii sp. nov.

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International Journal of Systematic Bacteriology 49 61 7 D. L. Williamson and others prokaryotes in Drosophila, leafhoppers and plants. Colloq plasma taxonomy and identification of the sex ratio organisms : INSERM 33,283-290. can they be cultivated? Yale J Biol Med 56, 583-592. Williamson, D. L. & Whitcomb, R. F. (1975). Plant mycoplasmas: Williamson, D. L., Hackett, K. J., Wagner, A. 1. & Cohen, A. 1. a cultivable spiroplasma causes corn stunt disease. Science 188, (1 989). Pathogenicity of cultivated Drosophila willistoni spiro- 10 18-1 020. plasmas. Curr Microbioll9, 53-56. Williamson, D. L., Whitcomb, R. F. & Tully, 1. G. (1978). The Williamson, D. L., Adams, J. R., Whitcomb, R. F., Tully, 1. G., spiroplasma deformation test, a new serological method. Curr Carle, P., Konai, M., Bov6, 1. M. & Henegar, R. B. (1997). Microbiol 1, 203-207. Spiroplasma platyhelix sp. nov., a new mollicute with unusual Williamson, D. L., Tully, J. G. & Whitcomb, R. F. (1979). Sero- morphology and genome size from the dragonfly Pachydiplax logical relationships of spiroplasmas as shown by combined longipennis. Int J Syst Bacteriol47, 763-766. deformation and metabolism inhibition tests. Int J Syst Williamson, D. L., Whitcomb, R. F., Tully, J. G. & 10 other authors Bacteriol29, 345-35 1. (1 998). Revised group classification of the genus Spiroplasma. Williamson, D. L., Steiner, T. & McGarrity, G. 1. (1983). Spiro- Int J Syst Bacteriol48, 1-12.

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