morphology ultrastructure helical nonhelical spiroplasma ·...

Vol. 142, No. 3JOURNAL OF BACTERIOLOGY, June 1980, p. 973-9810021-9193/80/06-0973/09$02.00/0

Morphology and Ultrastructure of Helical and NonhelicalStrains of Spiroplasma citri

ROD TOWNSEND,* JEREMY BURGESS, AND KITTY A. PLASKITTJohn Innes Institute, Norwich, Norfolk, England

Cells of the nonhelical strain of Spiroplasma citri underwent changes ofmorphology comparable to those which occurred in the normal helical strain.Cells of the nonhelical strain had the same ultrastructural features as helical cellsand released long flexible fibrils similar to those seen in other spiroplasmas.Nonhelical organisms showed an increased tendency to aggregate, forming. cellclusters of an unusual annular form. The cytoplasmic membrane of the nonhelicalstrain lacked a single protein present in all helical strains. Loss of helicityassociated with the senescence of spiroplasma cells was not accompanied by thedisappearance of this protein. Differences in colony morphology were shown tobe a consequence of motility, and a technique was developed which facilitatedthe identification of nonmotile organisms.

Numerous strains of spiroplasmas have beenisolated from plants and arthropods (7). All arehelical and motile except one strain of Spiro-plasma citri (ASP-1), which is nonhelical andconsequently nonmotile (22).Spiroplasma cultures often contain "medusa"-

like aggregates in which helices radiate from acentral mass of poorly defined bodies (3, 9).Strain ASP-1 is thought to show an increasedtendency to form aggregates, resulting in under-estimates of viable cells by methods based onviable counting (22). Scanning electron micros-copy has been used to compare the developmentof ASP-1 cultures with those of a helical strain(SP-A), with particular reference to the forma-tion of cell aggregates.The mechanisms by which spiroplasmas

maintain their helical shape in the absence of abacterial-type cell wall or are motile withoutintracellular or extracellular flagella (3) are un-known. Comparative studies of the properties ofhelical and nonhelical spiroplasmas may provideclues to the identity of these mechanisms. Par-tially purified membranes of strain ASP-1 lacka single protein ofmolecular weight 39,000 whichis present in all other isolates ofS. citri examined(21, 22), but the significance of this finding isunknown. As spiroplasmas age and growth con-ditions become suboptimal, the cells undergoseveral morphological changes including partialor complete loss of helicity and motility (3, 23).Changes in the protein composition of ageingspiroplasma membranes have been compared todetermine whether loss of helicity is associatedwith the disappearance of this protein.The nonhelical strain can be distinguished by

its colony morphology, which is of the "fried-

egg" form typical of many mycoplasmas. Thisresults from a combination of deep growth intothe agar and spreading over the surface (15).Spiroplasmas usually produce poorly definedcolonies with irregular central areas and periph-eral zones of variable diameters which have acoarse granular appearance. These features aremost obvious on soft agar (22) but can be influ-enced by conditions, prevailing on individualplates, which appear to inhibit motility. In thesecircumstances, colonies arising from motile andnonmotile organisms can be virtually indistin-guishable (R. Townsend, unpublished data).The structures ofSP-A and ASP-1 colonies havebeen compared by thin sectioning to confirmthat morphology is a consequence of motility,and a technique has been developed which en-sures consistent expression of the motile colonyform.

MATERIALS AND METHODSCulture of spiroplasmas. Nonhelical strain ASP-

1 of S. citri (National Culture Collection of PlantPathogenic Bacteria [NCPPB] no 3095) (22) and hel-ical strain SP-A (NCPPB 2565) isolated from a similarsource (5) were grown in modified complete sorbitolmedium (SMC) (17) supplemented with horse serum(20%, vol/vol; heat inactivated before use). Plates ofSMC solidified with agar (0.75 to 3%, wt/vol) weredried for 30 min, inoculated with a spiroplasma culture(0.1 ml), and incubated in sealed plastic bags or oversilica gel. Undried plates were incubated in a water-saturated atmosphere. Other inoculated plates wereoverlaid, after drying or after 24 h of incubation, withagar (0.75% in water at 45°C) to a depth of 2 to 3 mm.All cultures and plates were incubated at 32°C.Growth curves. SMC (5 ml) supplemented with

[2-'4C]thymidine (57 mCi mmol-', ca. 0.1 yCi ml-')973

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974 TOWNSEND, BURGESS, AND PLASKITT

were inoculated with an exponentially growing spiro-plasma culture (1%, vol/vol). Samples were removedat various time intervals, and growth was measured bydetermination ofthe quantity ofradioactive thymidineincorporated into DNA (4) and by counting colony-forming units (CFUs) (20).

Electrophoresis. Cells were harvested by centrif-ugation (15,000 x g for 30 min at 4°C) from cultures(100 ml) 48, 72, 96, and 144 h after inoculation withspiroplasmas (1%, vol/vol). Membranes were preparedby osmotic lysis (14), and their proteins were analyzedby polyacrylamide gel electrophoresis (6).Sample preparation and microscopy. Spiroplas-

mas were observed directly by dark-field microscopy,and colonies, unfixed or fixed and stained (18), wereexamined with a dissecting microscope. Cultures werefixed by the addition of an equal volume of glutaral-dehyde (7%, wt/vol) with sorbitol (7%, wt/vol) tomaintain a high osmotic pressure and prevent celldeformation (11). Unfixed organisms were negativelystained with methylamine tungstate (2%, wt/vol) orsedimented by centrifugation (20,000 x g for 15 min at4°C), and the resulting pellets were embedded in agar(1%, wt/vol) before being processed for thin sectioningand electron microscopy (12). Ruthenium red stainingwas used to test for the presence of capsular material(10). Preparations for scanning electron microscopywere made by adding 5 ml of spiroplasma culturediluted 1:2, 1:20, or 1:200 in sorbitol to an equal volumeof glutaraldehyde-sorbitol fixative in narrow centri-fuge tubes at the bottom of which were placed copperspecimen disks (1 cm diameter). After incubation atroom temperature, the organisms were sedimentedonto the disks by centrifugation (5,000 x g for 10 minat 20°C). The disks were washed in distilled water,and the spiroplasmas were dehydrated with increasingconcentrations of ethanol.

After dehydration, they were critical point driedfrom carbon dioxide, using amyl acetate as the inter-mediate solvent, and then coated with carbon andgold-palladium. Specimens were examined at 40 kV ina JEM 100B microscope fitted with an ASID scanningattachment (2).

Colonies were fixed in situ by allowing glutaralde-hyde-sorbitol solution to diffuse, overnight, into smallblocks of agar cut from inoculated plates and placedon pads of filter paper saturated with fixative.

RESULTSGrowth curves. Incorporation of radioactive

thymidine (Fig. la) revealed no latent phaseafter inoculation and a minimum genome dou-bling time of 6 h for both strains. When growthwas determined by CFUs (Fig. lb), SP-A andASP-1 showed latent phases of 16 and 24 h,respectively, but during exponential growth themaximum cell doubling time for both organismswas 3 h, and they reached titers in excess of 109CFUs/ml. Viability of both strains then declinedrapidly, falling below 106 CFUs/ml after 100 h,but incorporated radioactivity remained con-stant.

Cell morphology. During the first 24 h after

0 20 40 60 80 100Time after inoculation (h)

0 108

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0

.t

00

u 1060 20 40 60 80 100

Time after inoculation (h)FIG. 1. (a) Growth curves of S. citri strains SP-A

(0) and ASP-1 (0) at 32°C as measured by theincorporation of [2-14 Cithymidine. (b) Growth curvesof S. citri strains SP-A (a) and ASP-I (0) at 32°C asmeasured by CFUs.

inoculation, SP-A cultures contained short hel-ices of two or three turns, preceding cell division(Fig. 2a), and many small "knots" of organisms(Fig. 2b). These developed into larger aggregates2 to 5 [tm in diameter from which helices ra-diated (Fig. 2c). As cultures progressed into theexponential phase, many of these aggregates ap-peared to break up, liberating large numbers offreely motile spiroplasmas (Fig. 2d). By the endof exponential growth, only the larger aggregatesremained, most of which could be disrupted bygentle pipetting. As soon as viability began todecline, the pitch or tightness of the helicesbecame reduced; cells lengthened but failed todivide, and motility was eventually lost. After 72h, many of the cells had become partially orcompletely nonhelical, and loosely bound cellaggregates were again in evidence (Fig. 2e). Reg-ular constrictions began to appear along thelength of the filaments (Fig. 2f) until they weretransformed into chains of spherical bodies 0.2to 0.3 Itm in diameter.

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SPIROPLASMA MORPHOLOGY 975

FIG. 2. Scanning electron micrographs ofsamples from SP-A cultures after 24 h (a and b), 36 h (c), 47h (d),72 h (e), and 96 h (9). Arrows indicate constrictions formed before cell division. Bar, 2 mn.

Short "ropes" of laterally associated nonheli- aggregates persisted longer in ASP-1 culturescal cells predominated in ASP-1 cultures for the and were often more than 50 gm long (Fig. 3b),first 24 h. They were often joined end to end, but most eventually broke up to give smallforming closed loops or annuli (Fig. 3a). These clumps of loosely tangled organisms (Fig. 3c).

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FIG. 3. Scanning electron micrographs of samples from ASP-I cultures after 24 h (a), 36 h (b), 54 h (c), and96 h (d). Bar, 2 gm.

The filaments showed no evidence of helicity.They had constrictions indicative of dividingcells but were also branched with many budlikeprojections. None of the branches was helical.After 72 h, cells began to constrict along theirlength to form chains of spherical bodies (Fig.3d), and by 96 h it was impossible to distinguishASP-1 from SP-A cultures.

Effect of serum concentration. The for-mation ofaggregates in actively growing culturesof either strain depended on the quantity andsource of horse serum used. Lowering the con-

centration of horse serum to 10% (vol/vol) re-

duced their number and size markedly. Somebatches of serum caused increased aggregation,resulting in cell masses up to 100 ,um in diameterwhich formed a coarse precipitate in the culturevessels. Although cells on the outside of thesestructures were normal, those within the interiorhad lost their helical morphology and become

pleomorphic. These aggregates could not be bro-ken up by pipetting, and their occurrence re-

sulted in a reduction of more than 2 log units inthe peak CFU titer, although the quantity ofradioactive thymidine incorporated was almostunchanged.Ultrastructure. The trilaminar cytoplasmic

membranes of the two strains were structurallysimilar (Fig. 4a), and neither showed any evi-dence of capsular material which might causecells to adhere to each other. Negatively stainedASP-1 cells had barred structures, with a perio-dicity of 5 rum (Fig. 4b), and layers of surfaceprojections 5 nm long (Fig. 4c) similar to thosepreviously observed in S. citri cells (3). Lysedcells of the nonhelical strain also released 3.5-nm-diameter paired fibrils (Fig. 4d) which were

morphologically identical to those seen in otherspiroplasmas (19, 24, 25) and appeared equallyabundant in both strains.

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FIG. 4. Thin sectioned (a) and negatively stained (b through d) cells of ASP-I showing the trilaminarmembrane (a), barred structures (b), the layer of surface projections (arrowed) (c), and fibrils (d). Bars, 100nm.

Electrophoresis. A protein of molecularweight 39,000 was present in partially purifiedSP-A membranes and did not decline in concen-tration as cultures aged (Fig. 5). A correspondingband could not be detected in ASP-1 mem-branes.Colony morphology. Typical colonies of

both strains on 0.75% agar had a central core ofdeep growth into the agar and a layer of cellsabout 2 ,um thick spreading over the surface (Fig.6a and b). Cells in the body of a colony werepredominantly pleomorphic, but helical cellswere scattered throughout the agar in the im-mediate vicinity of SP-A colonies and were fre-quently observed more than 100 ,lm away fromthe center. There was no evidence of cell migra-tion away from ASP-1 colonies. The granularappearance of the peripheral zones around SP-A colonies was caused by the considerableamount of shallow growth below the surfacelayer and the presence of numerous satellitemicrocolonies composed of densely packed cells.There was very little subsurface growth aroundASP-1 colonies and no satellite colonies.Although differences in morphology were

most obvious on 0.75% agar, the diameters ofthe peripheral granular zones surrounding SP-Acolonies and development of satellites were veryvariable. These features were often more prom-inent around colonies at the edges of a platethan around those growing at its center. Some-times typical spiroplasma colonies with well-de-veloped satellites occurred next to colonies of amuch less granular appearance with no satel-lites. Such colonies could only be distinguishedfrom those of ASP-1 after careful examination.

Colonies of both strains were smaller, theamount of growth into the agar was reduced,and the distance which spiroplasmas migratedwas progressively lessened the higher the con-centration of agar tested. Above a concentrationof 2% (wt/vol), only surface growth was appar-ent, the fried-egg morphology was lost, and thetwo strains were indistinguishable (Fig. 7).

In a water-saturated atmosphere, both strainsproduced similar large fried-egg colonies 1 to 2mm in diameter with extensive surface growth.Under these conditions, the surface layer of SP-A colonies contained numerous helical cells or-ientated at right angles to the agar surface (Fig.

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SP-A ASP-1I .I.

allowed to develop for 24 h before overlaying,there was no significant loss of CFU.

DISCUSSION

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1234 1234FIG. 5. Electrophoreticpatterns ofSP-A andASP-

I partially purified membranes after 48 h (1), 72 h (2),96 h (3), and 144 h (4) showing the absence of 39,000-molecular-weight protein (arrowed) from ASP-I.

8a), whereas ASP-1 colonies contained predom-inantly filamentous cells lying parallel to thesurface of the agar (Fig. 8b). Plates incubatedunder drying conditions showed tiny colonies orno colonies at all.

Overlaying with agar consistently caused SP-A cells to form typical spiroplasma colonies ofan even size and with prominent satellites.Strain ASP-1 produced thick, biconvex colonieslacking peripheral zones and satellites, whichwere easily distinguished from those of the mo-tile strain (Fig. 9). Plates could be stained in theusual way (18), and the overlay prevented colo-nies of ASP-1 being lost during washing. Countsof CFUs made on these plates were sometimeslower than those made on plates which had notbeen overlaid. This was apparently due to thedeath of a few organisms caused by the warm

agar or the loss of cells which floated off theplate surface before the agar set. If colonies were

The mode of cell division in both strains ap-peared to be similar, but the frequency ofbranching and budding of ASP-1 cells suggestedthat processes such as cell elongation and mem-brane assembly were impaired. Apart from lossof helicity, all other changes in the morphologyof SP-A were paralleled in ASP-1, including theformation of spherical bodies. These have beendescribed as viable sporelike cells because theyappeared to extrude one or more thin, opticallyhomogeneous filaments when negatively stainedwithout prior fixation (9). The concept of thesestructures as "spores" is incompatible with therapid decline ofviability associated with culturesin which they predominate. Furthermore, al-though spiroplasmas may contain as many asfive complete genomes (S. A. Field, Sixty-Sev-enth Annual Report ofthe John Innes Institute,1975), each organism can produce up to 80 spher-ical bodies without further DNA replication, sothat the majority of "spores" would containincomplete genomes.The formation of cell aggregates resulted in

anomalies between measurements of growthbased on DNA replication and CFUs. The lagphase indicated by viable counts reflected thefailure of cells to separate after division, leadingto the formation of aggregates. The longer du-ration of this phase in ASP-1 cultures was anindication of their increased tendency to aggre-gate. The annular structure of these aggregateswas most probably a consequence of the ifia-mentous morphology of the organisms. Duringthe phase of exponential growth, the apparentcell doubling time as measured by CFUs wasaffected by the large numbers of free cells re-leased as aggregates broke up.The nature of the serum used to supplement

growth media is an important factor in deter-mining the extent to which cells aggregate. Trep-onemes grown in media containing serum canalso form cell aggregates, and it has been shownthat this is due to the presence of antibodies tothese organisms elicited in the donor animal byrelated bacteria which are part of the normalflora (J. Pillot, Ph.D. thesis, University of Paris,Paris, France, 1965). Such an explanation seemsunlikely in the case of spiroplasmas which arenot known to infect vertebrates naturally. Fur-thermore, Mycoplasma pneumoniae also formslarge aggregates known as "spherules," whichare composed of tightly packed cells, but these

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FIG. 6. Thin sections through small colonies of SP-A (a) and ASP-I (b) growing on SMC solidified with0.75% agar, showing the normal spiroplasma morphology and the differences resulting from the absence ofmnotility in strain ASP-I. Bar, 5 pm.

FIG. 7. Thin section through a colony of SP-A growing on SMC solidified with 2%o (wt/vol) agar andshowing the absence ofgrowth into the agar. Bar, 5 ,um.

VOL. 142, 1980

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FIG. 8. Thin sections through colonies growing in a water-saturated atmosphere, showing helical SP-Acells orientated at right angles to the agar surface (a) and filamentous ASP-I cells lying parallel to the agarsurface (b). Bars, 2.5 ,tm.

a

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b

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FIG. 9. Stained colonies ofSP-A (a) and ASP-I (b)growing under an overlay, showing the differences inmorphology. Bar, 0.5 mm.

can arise in cultures supplemented with agammahorse serum (1).

It has been demonstrated that the myco-plasma Acholeplasma laidlawii is capable ofbinding soluble proteins, including bovine serumalbumin, to the cell membrane (16). Therefore,it seems possible that spiroplasmas could be-come bound together through the intermediaryof a soluble plasma protein. However, since thebinding effect was enhanced at acid pH values,it is more likely that such a mechanism couldaccount for the loosely bound cell clumps formedas cultures aged and became acidic.The pleomorphic organisms occurring in large

aggregates resembled spiroplasma cells in thecenter of colonies. This morphology may beindicative of senescence, which would be con-trary to the changes that occur in liquid cultures.Since alterations in the composition of thegrowth medium can induce similar changes (8),it seems probable that cells become pleomorphicbecause of the depletion of lipids or other nutri-ents. This is supported by the observation thatspiroplasmas remained helical on plates with a

layer of surface moisture, which facilitated thediffusion of nutrients.A hypothesis to account for spiroplasma hel-

icity has been proposed based on the associationof helically wound 3.5-nm fibrils with the innersurface of the cytoplasmic membrane (13). Ten-sing and compressing these fibrils in the correctsequence could provide forces capable of gener-ating a helix and producing rotary movement.The presence of fibrils in the nonhelical straindoes not invalidate this hypothesis. To bring theforces generated by contraction to bear upon thecell, each fibril must be securely anchored, atmany points along its length, to the cytoplasmicmembrane. Contraction of fibrils without ade-quate structural association with the membranecould result in erratic flexing movements similarto those produced by ASP-1 cells (22). It there-fore seems plausible that the 39,000-molecular-weight protein may be part of the structureconnecting fibril elements to the membrane.Colony morphology is a direct consequence of

motility. The formation of typical spiroplasmacolonies depends on the ability of organisms topenetrate the agar surface and to swim throughthe agar away from the parent colony. Agarconcentration is, therefore, a prime determinantof colony morphology, but local conditions atthe point of inoculation, such as the amount ofsurface moisture, can markedly affect subse-quent colony development. Overlaying plateswith agar eliminates these variations and en-courages cell migration. This technique shouldprove of value in the field of spiroplasma ge-netics, since it enables nonmotile mutants to be

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selected with relative ease. Overlaying inevitablylifts some cells off the top of developing colonies,which can remain viable in the agar, or evenform small colonies utilizing nutrients diffusingthrough the agar. Therefore, isolates selected bythis method must be cloned to eliminate possiblecontamination from this source.

LITERATURE CITED

1. Boatman, E. S., and G. E. Kenny. 1971. Morphologyand ultrastructure of Mycoplasma pneumoniae spher-ules. J. Bacteriol. 106:1005-1015.

2. Burgess, J., P. J. Linstead, and V. E. L. Fisher. 1977.Studies on higher plant protoplasts by scanning electronmicroscopy. Micron 8:113-122.

3. Cole, R. M., J. G. Tully, T. J. Popkin, and J. M. Bove.1973. Morphology, ultrastructure, and bacteriophageinfection of the helical mycoplasma-like organism (Spi-roplasma citri gen. nov., sp. nov.) cultured from "Stub-bom" disease of citrus. J. Bacteriol. 115:367-386.

4. Daniels, M. J. 1969. Lipid synthesis in relation to the cellcycle ofBacillus megaterium KM and Escherichia coli.Biochem. J. 115:697-701.

5. Daniels, M. J., P. G. Markham, B. M. Meddins, A. K.Plaskitt, R. Townsend, and M. Bar-Joseph. 1974.Axenic culture of a plant pathogenic spiroplasma. Na-ture (London) 244:523-524.

6. Daniels, M. J., and B. M. Meddins. 1973. Polyacrylam-ide gel electrophoresis of mycoplasma proteins in so-dium dodecyl sulphate. J. Gen. Microbiol. 76:239-242.

7. Davis, R. E. 1979. Spiroplasmas: newly recognised ar-thropod-borne pathogens, p. 451-484. In K. Maramo-rosch and K. F. Harris (ed.), Leafhopper vectors andplant disease agents. Academic Press, Inc., New York.

8. Freeman, B. A., R. Sissenstein, T. T. McManus, J. E.Woodward, I. M. Lee, and J. B. Mudd. 1976. Lipidcomposition and lipid metabolism of Spiroplasma citri.J. Bacteriol. 125:946-954.

9. Fudh-Allah, A. E.-S. and E. C. Calavan. 1974. Cellularmorphology and reproduction of the mycoplasma-likeorganism associated with citrus stubborn disease. Phy-topathology 64:1309-1313.

10. Howard, C. J., and R. N. Gourlay. 1974. An electron-microscopic examination of certain bovine mycoplas-mas stained with ruthenium red and the demonstrationof a capsule on Mycoplasma dispar. J. Gen. Microbiol.83:393-398.

11. Lemcke, R. M. 1972. Osmolar concentration and fixation

of mycoplasmas. J. Bacteriol. 110:1154-1162.12. Markham, P. G., R. Townsend, M. Bar-Joseph, M. J.

Daniels, A. Plaskitt, and B. M. Meddins. 1974. Spi-roplasmas are the causal agents of citrus little-leaf dis-ease. Ann. Appl. Biol. 78:49-57.

13. Razin, S. 1978. The mycoplasmas. Microbiol. Rev. 42:414-470.

14. Razin, S., M. Hasin, Z. Ne'eman, and S. Rottem. 1973.Isolation, chemical composition, and ultrastructural fea-tures of the cell membrane of the mycoplasma-likeorganism Spiroplasma citri. J. Bacteriol. 116:1421-1435.

15. Razin, S., and 0. Oliver. 1961. Morphogenesis of myco-plasma and bacterial L-form colonies. J. Gen. Microbiol.24:225-237.

16. Rottem, S., M. Hasin, and S. Razin. 1973. Binding ofproteins to mycoplasma membranes. Biochim. Biophys.Acta 298:876-886.

17. Saglio, P., D. Lafleche, C. Bonissol, and J. M. Bove.1971. Isolement, culture et observation au microscopeelectronique des structures de type mycoplasma asso-ciees a la maladie du stubborn des agrumes et leurcomparison avec les structures observees dans le cas dela maladie du Greening des agrumes. Physiol. Veg. 9:569-582.

18. Scriba, M. 1968. Experiments to eliminate mycoplasmasfrom tissue cultures by means of antibiotics. Z. Med.Microbiol. Immunol. 154:267-276.

19. Stalheim, O. H. V., A. F. Ritchie, and R. F. Whitcomb.1978. Cultivation, serology, ultrastructure and virus-likeparticles of spiroplasma 277F. Curr. Microbiol. 1:365-370.

20. Townsend, R. 1976. Arginine metabolism by Spiro-plasma citri. J. Gen. Microbiol. 94:417-420.

21. Townsend, R. 1978. Non-helical isolates of Spiroplasmacitri. Zentralbl. Bakteriol. Parasitenkd. Infektionskr.Hyg. Abt. 1 Orig. Reihe A 241:223-224.

22. Townsend, R., P. G. Markham, K. A. Plaskitt, and M.J. Daniels. 1977. Isolation and characterisation of anon-helical strain of Spiroplasma citri. J. Gen. Micro-biol. 100:15-21.

23. Whitcomb, R. F., and D. L. Williamson. 1975. Helicalwall-free prokaryotes in insects: multiplication andpathogenicity. Ann. N.Y. Acad. Sci. 266:260-275.

24. Williamson, D. L. 1974. Unusual fibrils from the spiro-chete-like Sex Ratio Organism. J. Bacteriol. 117:904-906.

25. Williamson, D. L., and R. F. Whitcomb. 1974. Helical,wall-free prokaryotes in Drosophila, leafhoppers andplants. Colloq. Inst. Natl. Sante R. Med. 33:283-290.

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