INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,Apr. 1987, p. 106-115 Vol. 37, No. 2 0020-7713/87/020106-10$02.OO/O Copyright 0 1987, International Union of Microbiological Societies

Spiroplasma phoeniceum sp. nov. a New Plant-Pathogenic Species from Syria C. SAILLARD,' J. C. VIGNAULT,l J. M. BOVE,l* A. RAIE,2 J. G. TULLY,3 D. L. WILLIAMSON,4 A. FOS,' M. GARNIER,l A. GADEAU,l P. CARLE,l AND R. F. WHITCOMB' Laboratoire de Biologie Cellulaire et Mole'culaire, Institut National de la Recherche Agronomique et Universitk de Bordeaux II, 33140 Pont de la Maye, France'; Agricultural Services, Lattakia, Syria2; Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Center, Frederick, Maryland 21 7013; Department of Anatomical Sciences, Health Sciences Center, State University of New York, Stony Brook, New York 11 7944; and Insect Pathology Laboratory, Plant Protection Institute, Beltsville Agricultural Research Center, U.S.Department of Agriculture, Beltsville, Maryland 20705'

Sixteen isolates, recovered over a 2-year period from symptomatic periwinkle plants (Catharanthus roseus) collected in eight different locations in Syria, were compared with other established Spiroplasma species or serogroups. Serological analysis of selected representatives of the new isolates revealed sharing of some antigenic components with several currently classified within subgroups of group I of the genus. Strain P40T was selected as the type strain and examined, meeting the criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on the of Mollicufes. The organism was shown to belong to the class Mollicufes by its morphology, ultrastructure of its limiting membrane, colony characteristics, and filtration patterns. The helicity and motility of the cells indicated its placement within the family Spiroplasmafaceae. Although some serological cross-reactions could be observed with representatives of group I subgroups, strain P40T could be readily distinguished from other plant or insect pathogenic spiroplasmas in subgroup 1-1 (Spiroplasma cif7-2'9, subgroup 1-2 (S. melliferum), or subgroup 1-3 (S. kunkeliq and from spiroplasmas assigned to subgroups 1-4 through 1-7 and groups I1 through XI. Cholesterol was required for growth. Glucose was fermented, and arginine was hydrolyzed. The base composition (guanine plus cytosine) of the deoxyribonucleic acid of strain P40T was found to be 26 mol%. Deoxyribonucleic acid-deoxyribonucleic acid hybridization comparisons between strain P40T and other subgroup I representa- tives revealed approximately 60% relatedness to S. cifri and S. kunkelii and 50% relatedness to S. mellverum. Experimental transmission of two of the new isolates (P40T and P354) occurred through inoculation of spiroplasma broth cultures into (Macrosfelesfascifrons), multiplication of the organism in the insects, and subsequent transmission of the organism by insect feeding on aster or periwinkle plants. The organism was also successfully recovered from broth cultures of symptomatic tissues of experimentally infected periwinkle plants, thus fulfilling Koch's postulates. We propose that such strains be named Spiroplasma phoeniceum. Strain P40T has been deposited 'in the American Type Culture Collection (= ATCC 43115T)

Although many Spiroplasma species have been isolated (DNA) probe of this strain was tested against S. citri DNA, during the last 10 years from insects and plants, only two- a value of 60% homology was obtained, indicating that (47), the etiological agent of stub- subgroup 1-8 spiroplasmas, like other group I spiroplasmas, born disease (38), and S. kunkelii (60), the spiroplasma that share patterns of partial relatedness. Taxonomic difficulties causes corn stunt disease (11, 68)-are plant pathogenic. posed by partial relationships of group I spiroplasma strains Citrus stubborn disease is widespread in the Mediterranean (57) were discussed by the International Committee on region as a result of the natural dissemination of S. citri by Systematic Bacteriology Subcommittee on the Taxonomy of leafhoppers (2, 24). In surveys designed to demonstrate (30, 31). Pending results of studies on new natural transmission of this disease in Syria, 2-month-old, spiroplasmas from insects and plants, a conservative ap- healthy periwinkles (Catharanthus roseus) were exposed proach to the description of subgroups was recommended. during summer months in the vicinity of orchards with high The Subcommittee in 1982 proposed (32) that subgroups rates of stubborn disease infection. In October, many peri- might be elevated to species status under certain conditions, winkles showed symptoms typical of mycoplasma-like orga- as follows: (i) required steps in the minimal-standards doc- nism (MLO) or S. citri infection or both (3). The presence of ument (29) must be fulfilled; (ii) DNA-DNA reassociation S. citri was confirmed in these symptomatic plants by two between the candidate subgroup and all other subgroups techniques-culturing of the organism in standard liquid must be demonstrated to be less than 70%; (iii) the ecology medium and the enzyme-linked immunosorbent assay of the organism must be studied, and its hosts must be (EIJSA) (2, 6, 48). However, with eight yellowed periwin- identified; (iv) the subgroup should be of agricultural impor- kles, although spiroplasma cultures were obtained, ELISAs tance or serve as a basic microbiological model; and (v) the for S. citri were negative. Serological analysis of these subgroup should represent a cluster of homogeneous strains, isoliates (51) showed that they represented a new subgroup as assessed by techniques such as serology and polyacryl- (1-8) of Spiroplasma group I. Strain P40T was chosen as a amide gel electrophoresis (PAGE). This proposal was representative. When a labeled deoxyribonucleic acid prompted by the demonstration of clusters of relative geno- typic homogeneity in several mollicute species (46). Whether a particular set of data fulfilled the conditions was left to the * Corresponding author. judgment of referees and editors.

106 VOL. 37, 1987 SPIROPLASMA PHOENICEUM SP. NOV. 107

TABLE 1. Summary of periwinkles naturally infected with S. cifri or S. phoeniceum in 1983 and 1984 in Syria Periwinkle Location Period of exposure designation Infecting agent(s) Wizan 108 June 1984 S. phoeniceum El Annadeh project Nursery 111 3 14 17 July to 9 August 1983 S. cifri 62 and 43 July 1984 S. citri 33 September 1983 to June 1984 S. phoeniceum Sweet orange mother tree plot 19 September 1983 to June 1984 S. phoeniceum + MLO (virescence) 47 July 1984 S. phoeniceum + MLO (vire scence) El Annadeh government nursery 36 September 1983 to June 1984 S. phoeniceum El Annadeh, Fidio 103 July 1984 S. phoeniceum + MLO (no virescence) El Annadeh, Sidawi orchard 41 17 July to 18 October 1983 S. phoeniceum P40T 17 July to 18 October 1983 S. phoeniceum 42 September 1983 to June 1984 S. citri JablC Citrus Experiment Station 37, 341, and P354 17 July to 23 October, 1983 S. phoeniceum Tartous Center of Agriculture 198 and 199 17 July to 19 October 1983 S. phoeniceurn Amrit Citrus Office 263 17 July to 19 October 1983 S. phoeniceum 24 September 1984 S. phoeniceum

In this report, we summarize experimental evidence that 68]), tick spiroplasma strain 277F (= ATCC 29761) (sub- strain P40T and related strains represent a cluster of group 1-4 [8, 53]), strain LB-12 (= ATCC 33649) (subgroup spiroplasmas for which the species requirements have been 1-5 [36]), Maryland flower spiroplasma strain M55 (= ATCC fulfilled, with respect to both the Subcommittee proposal for 33502) (subgroup 1-6 [64]), COCOSspiroplasma strain N525 the elevation of subgroups to species status (32) and the (= ATCC 33287) (subgroup 1-7 [21]), uncultivated strain minimal standards for species descriptions of members of WSRO of sex ratio organism (group I1 [66]), S. the class Mollicutes proposed in 1979 by the Subcommittee Jloricola OBMG (= ATCC 33221) (group I11 [13, 19]), S. apis (29). We therefore formally propose that strain P40T and B31T (= ATCC 33834T) (group IV [42,43]), S. apis PPSl (= related strains be recognized as a new species in the genus ATCC 33450) (group IV [40]), S. mirum SMCAT (= ATCC Spiroplasma. 29335T) (group V [58,59]), Zxodes spiroplasma strain Y32 (= ATCC 33835) (group VI [56]), Monobia spiroplasma strain MATERIALS AND METHODS MQ-1 (= ATCC 33825) (group VII [14, 62]), syrphid spiro- plasma strain EA-1 (= ATCC 33826) (group VIII [14, 62]), Plant material. Two-month-old, healthy periwinkle plants Cotinus spiroplasma strain CN-5 (= ATCC 33827) (group IX (C. roseus) were exposed to natural transmission during [15, 61]), Aedes spiroplasma strain AES-1 (= ATCC 35112) various months in 1983 to 1984 at different locations near or (group X [61]), and Monobia spiroplasma strain MQ-4 (= within stubborn disease-affected citrus orchards of the Syr- ATCC 35262) (group XI [61]). ian coastal area between Lattaquia and Tartous (see Table Biochemical tests. Substrate tests were performed in 1% 1). In October, the periwinkles were examined for symptom bovine serum fraction broth by procedures described earlier expression and were tested by ELISA and culture for the (1, 16). Phosphatase activity, film-and-spot reaction, and presence of S. citri. hemadsorption tests were performed on solid medium (SP-4 Culture media and cultivation procedures. Strain P40T and broth with 0.8% Noble agar) by previously described tech- other subgroup 1-8 strains were cultivated in M1A (33) or niques (1, 7, 23, 37). SP-4 (58) medium. For spiroplasma maintenance, cultures Serological tests. Hyperimmune antiserum was prepared were passed every 2 or 3 days as a 10% inoculum added to against strain P40T as described previously (69). Disk growth fresh medium. To isolate organisms from periwinkles (6), we inhibition tests (17, 63) were performed with spiroplasmas cut leaf midribs into small pieces and soaked them in 3 ml of grown in M1D medium (60). Antigens grown in broth were liquid medium. After 1h at room temperature, the broth was usually diluted to about lo5 color-changing units per ml. filtered through 450-nm-pore filters, 0.3 ml of the sterile Techniques for deformation tests, inhibition filtrate was added to 3 ml of penicillin-free M1A or SP-4 tests, and ELISAs have already been published (48, 49, 67, medium, and the mixture was incubated at 32°C. After about 69). Most metabolism inhibition test antigens were prepared 10 days, indicator color change was observed, and 0.3 ml of from cultures grown for 24 h in SP-4 broth; most test the culture was transferred to 3 ml of fresh medium. Agar procedures were performed in this medium. However, the colonies of subgroup spiroplasmas were obtained by plating metabolism inhibition test antigen for S. kunkelii E275T was cultures on M1A or SP-4 medium supplemented with 0.8% from a culture grown in M1A broth, and the test procedures Noble agar (soft agar) (Difco Laboratories). were performed in this medium. Although most spiroplasmas used in comparative tests Filtration studies. Broth cultures of strain P40T in SP-4 were cultivated in M1A medium, S. mirum (group V) and medium that had been incubated for 4 days at 32°C were strain Y32 (group VI) were grown in SP-4 medium (58). passed through a series of membrane filters with graded pore Sources of other spiroplasmas. Other spiroplasmas were diameters (450, 300, 220, and 100 nm). Each filtrate was compared with spiroplasma strain P40T. These included S. diluted in a series of 10-fold dilutions in SP-4 medium, and all citri Mar0c-R8A2~(= ATCC 2755ST) (subgroup 1-1 [5, 47]), tubes were incubated at 32°C. After 7 to 14 days, the tubes S. melliferum BC-3T (= ATCC 33219T) (subgroup 1-2 [12, were examined for color change. The last dilution in each 16]), S. kunkelii E275T(= ATC 29320T) (subgroup 1-3 [ll,60, series that showed helical cells and a color change was 1018 SAILLARD ET AL. INT. J. SYST.BACTERIOL.

FIG. 1. Electron micrograph of strain P40T after transfer from liquid medium (MlA) to a collodion membrane. The spiroplasma was cultured at 32°C for 8 days. p, Micrometer. noted. The results were expressed in color-changing units and 37°C. The incorporation of 32Pinto spiroplasma nucleic per milliliter. acids was determined from the radioactivity of the insoluble Morphology. Cultures of strain P40T were observed by trichloracetic acid precipitate from 100 pl of culture. A 5-ml dark-field microscopy (55) and by electron microscopy after quantity of 15% trichloroacetic acid was used, and the transfer to collodion membranes (25, 26). Thin sections of precipitate was filtered on glass fiber filters (Whatman GFA) pelleted organisms were obtained as described previously and washed with 20 ml of 10% trichloroacetic acid. (47). All plant tissues to be examined by electron microscopy Phospholipids were removed with 5 ml of a 50% ethanol- were fixed for 4 h in 2.5% glutaraldehyde in cacodylate 50% ether mixture and then with 5 ml of ether alone. The buffer, postfixed in 1% osmium tetroxide for 2 h, dehydrated radioactivity was counted in a scintillation spectrometer in alcohol, and embedded in Epon 812. (Intertechnique SC30). Temperature requirements. Temperature requirements for Sterol requirements. The direct broth culture method for growth were determined by the incorporation of 32P into the cholesterol test was performed as described earlier (45). nucleic acids of strain P40T (47). A 100-ml quantity of PAGE of cell proteins. Techniques for the determination of penicillin-free M1A medium containing 500 pCi of 32P was patterns of cell proteins in one-dimensional PAGE have been inoculated with 2 ml of an exponentially growing culture. published previously (41). This seeded medium was divided and incubated at 28, 32, Genomic analysis. Techniques used for extraction and

FIG. 2. Electron micrograph of a thin section of strain P40T showing the unit membrane surrounding the cell (arrows). p, Micrometer. VOL. 37, 1987 SPIROPLASMA PHOENICEUM SP. NOV. 109

ably infected with two agents: the subgroup 1-8 spiroplasma and an MLO (3). Strains P40T and P354 were obtained from periwinkles exposed in 1983 in the Sidawi orchard at El Annadeh and in the Citrus Experiment Station at JablC, 2 8°C respectively. Experimental transmission. Although the natural insect vectors and plant reservoirs have not been discovered, subgroup 1-8 spiroplasmas have been shown to incite disease 32OC in plants (R. F. Whitcomb, A. L. Hicks, E. A. Clark, C. Saillard, A. Fos, J. G. Tully, and J. M. BovC, manuscript in preparation). The Macrosteles fascifrons , a nat- ural vector (35) of the aster yellows MLO (39) and an experimental vector of S. citri (44, 65; M. El-Bolok, Ph.D. thesis, University of Cairo, Giza, Egypt, 1980), was injected with cultivated organisms of strain P40T and strain P354. The spiroplasmas multiplied readily in this insect, with a sugges- tion of an adverse effect on the leafhopper. The insects subsequently transmitted the spiroplasmas to aster (Cal- listephus chinensis) and Madagascar periwinkle. In aster, symptoms resembled, but were distinguishable from, symp- toms of S. citri infections and included yellowing and mal- formation of the leaves, rosetting of the plants, and dwarfing of the flowers. The flowers were dwarfed but not virescent, as in diseases induced by certain MLOs (3,35). In C.roseus, spiroplasmas could be cultivated from the infected plants and were shown to be serologically indistinguishable from subgroup 1-8 organisms. Thus, Koch's postulates were ful- 0 24 48 72 #I 120 144 hr. filled. FIG. 3. Growth of strain P40T at different temperatures in peni- Morphological and cultural properties. Primary cultures of cillin-free M1A medium, as measured by 32P incorporation into strain P40T and 15 other subgroup 1-8 strains were obtained nucleic acids. after 8 days in antibiotic-free SP-4 and M1A broth media at 32°C. Cultures examined by dark-field microscopy and by determination (by buoyant density and melting point) of the electron microscopy (Fig. 1) contained many long, thin, guanine-plus-cystosine (G+ C) content (in moles percent) of helical cells. Thin sections revealed, as is typical for the the DNA of strain P40T have been previously described (9, genus (18), only a single unit membrane surrounding the cell 10). The two DNA-DNA hybridization techniques (S1 nu- (Fig. 2). During early passages, long intervals were required clease and hydroxyapatite chromatography) used to assess between transfers (10 days), and electron-microscopic ex- relatedness between the DNA of strain P40T and the DNAs amination revealed many virions of the SpV3 and SpVl of all the other strains were as described before (20), except types (18). The presence of free virions was associated with that the hybridization reaction was carried out in the absence the decreased growth rate of strain P40T. When inoculated at of formamide and at 66°C for 16 h. The melting temperature a 10% inoculum level every 2 days, strain P40T and other (T,) of the DNA hybrids was determined by the strains eventually grew rapidly and, as the time required for hydroxyapatite technique. The percentage of mismatched passages declined, virus particles decreased in number or base pairs was calculated from the T, by the method of became undetectable. Hyman et al. (28). Detection of strain P40T in plants. The ELISA, performed TABLE 2. Sterol requirement test for strain P40T on crude plant extracts by techniques described earlier (48, Amt of cell protein 49), was used for the detection of strain P40T in plants (mg)" in expt: infected by grafting with shoots of naturally infected peri- Medium winkles. 1 2 Serum free (B medium) <0.02

TABLE 3. Growth inhibition tests with strain P40T - Diam (mm) of inhibition zone around disk saturated with antiserum" P40T antiserum Group Strain P40T antigen vs Homologous vs heterologous heterologous reaction antisera antigens 1-1 S citri Mar0c-R8A2~ 11 6 16 1-2 S. melliferum BC-3T 12 2 12 1-3 S. kunkelii E275T 12 6 18 1-4 277F N N 14 1-5 LB-12 4 N 18 1-6 M55 7 N 13 1-7 N525 N N 11 1-8 P40T 17 I1 WSRO ND ND ND I11 S.floricola OBMG 2 N 6 IV S. apis B31T N N 5 V S. mirum SMCAT N N 13 VI Y32 N N 16 VII MQ-1 N N 6 VIII EA-1 N N 10 IX CN-5 N N 8 x AES-1 N N 5 XI MQ-4 N N 6

a N, No zone of inhibition; ND, not done.

Strain P40T grew optimally in M1A medium at 32°C (Fig. As with both S. citri (54) and other named group I species 3), reaching titers of los to 10" after 4 to 14 days. Little or no (16,47,60), when both substrates were present, the medium growth occurred at 37°C. On solid medium containing 0.8% first became acid but later, as arginine was catabolized, Noble agar, classical "fried-egg" colonies appeared after 7 shifted to an alkaline pH. Strain P40T did not hydrolyze urea. days of incubation at 32°C. The tests for phosphatase reaction and film-and-spot re- Filtration studies. A broth culture of strain P40T containing sponse were positive. Colonies of strain P40T grown on soft 108 color-changing units per ml was filtered through a series agar hemadsorbed guinea pig erythrocytes. of graded membrane filters. The numbers of color-changing Sterol requirements. Significant growth of strain P40T was units per milliliter after filtration through membrane filters not observed in a serum-free medium or when supplements with 450-, 300-, and 220-nm-pores were lo8, lo6, and lo6, of albumin, palmitic acid, and Tween 80 were added (Table respectively. The organism did not pass through 100-nm- 2). The addition of cholesterol at levels of 5 to 20 pg/ml to the pore filters. base medium (D medium, Table 2) generally increased the Biochemical and biological properties. Strain P40T fer- growth of the organisms, indicating a sterol requirement for mented glucose and hydrolyzed arginine, the latter response growth. occurring whether or not glucose was present in the medium. Serological tests. Serum prepared against the P40T strain

TABLE 4. Metabolism inhibition tests with strain P40T Titer" P40T antigen vs P40T antiserum Group Strain Homologous heterologous vs heterologous reaction antisera antigens

~ ~~~ ~ 1-1 S. citri Mar0c-R8A2~ 468 4,374 > 117,000 1-2 S. melliferum BC-3T 1,458 486 >117,000 1-3 S. kunkelii E275T 486 486 39,000 1-4 277F 162 N 39,000 1-5 LB-12 4,374 4,374 >1 17,000 1-6 M55 18 N 13,000 1-7 N525 162 18 39,000 1-43 P40T >117,000 I][ WSRO 54 ND ND II[I S. Jloricola OBMG 54 N 13,000 IV S. apis B31T 18 N 13,000 V S. mirum SMCA~ 18 N 13,000 \,I Y 32 N N 4,374 v I1 MQ-1 18 N >117,000 VIII EA-1 18 N 39,000 IX CN-5 N N 4,374 X AES-1 N N 4,374 XI MQ-4 18 N 4,374

'I Reciprocal of the highest antiserum dilution which inhibited metabolism. N, Titer of less than 1:18; ND, not done. VOL. 37, 1987 SPIROPLASMA PHOENICEUM SP. NOV. 111

TABLE 5. Deformation tests with strain P40T

~~ Titer"

Group Strain P40T antigen vs P40T antiserum heterologous vs heterologous Homologous antisera antigens reaction I- 1 S. citri Mar0c-RgA2~ 20 80 2,500 1-2 S. rnelliferurn BC-3T 1,280 80 10,240 1-3 S. kunkelii E27jT 320 640 20,480 1-4 277F 20 N 5,120 1-5 LB-12 2,560 160 5,120 1-6 M55 N N 1,280 1-7 N525 20 N 10,240 1-8 P40T 10,240 I1 WSRO N N 2,560 I11 S. JEoricola OBMG N N 10,240 IV S. apis B31T N N 1,280 V S. mirurn SMCAT N N 5,120 VI Y 32 N N 2,560 VII MQ-1 N N 1,280 VIII EA- 1 N N 1,280 IX CN-5 N N 640 X AES-1 N N 640 XI MQ-4 N N 640

~~ ~ Reciprocal of the highest antiserum dilution in which one-half the spiroplasmas are free and motile and one-half are deformed. N, Titer of less than 1:20. gave a zone of homologous growth inhibition with a diameter PAGE. Patterns of strain P40T obtained from one- of 17 mm. In the heterologous reaction, growth inhibition dimensional PAGE of cell proteins (Fig. 4) were significantly was observed only with S. citri (Mar0c-R8A2~),S. mel- different from patterns of the seven other members of group liferurn (BC-3T),and S. kunkelii (E275T);the zones of growth I. The homogeneity of the subgroup 1-8 organisms was inhibition were, respectively 6, 2, and 6 mm (Table 3). The assessed by comparing the one-dimensional protein pattern growth of strain P40T was inhibited only by antisera directed of eight strains. The patterns of six isolates obtained in 1983 against other group I spiroplasmas; however, sera against (Fig. 5a) were almost identical, and the patterns of four strain 277F (subgroup 1-4) and strain N525 (subgroup 1-7) isolates obtained in 1984 were very similar to that of the P40T failed to produce growth inhibition zones (Table 3). isolate from 1983 (Fig. 5b). Metabolism inhibition (Table 4) and deformation (Table 5) Genomic analysis. The G + C content,of the DNA of strain tests gave essentially the same results as growth inhibition, P40T was determined from the buoyant density (obtained by in that only group I spiroplasmas showed serological relat- equilibrium centrifugation in CsCl in five independent anal- edness with strain P40T. When P40T antiserum was tested yses) or from the Tm (assayed spectrophotometrically in four against heterologous antigens, S. citri (Mar0c-RgA2~)and experiments). The G+C content was found to be 26 2 1 strain LB-12 (subgroup 1-5) exhibited the greatest related- mol%, as determined from the buoyant density and the Tm. ness. When P40T antigens were tested against heterologous This value is generally characteristic of other group I antisera, S. melliferum (BC-3T)and strain LB-12 (subgroup spiroplasmas (4, 5, 34). 1-5) were found to have the greatest serological relationship DNA-DNA hybridizations. Significant hybridization of with P40T. These data confirm the distinctiveness of strain DNA from strain P40T and DNAs from other members of P40T from all existing groups, subgroups, and species. They group I was observed (Table 6). Other groups showed no demonstrate that strain P40T represents a serological sub- significant relatedness. Approximately 60% hybridization group that is distinguishable from other subgroups of group I, from S.floricola , S. apis , and S, mirum, and from all other currently recognized spiroplasma groups (I1 to XI).

FIG. 4. One-dimensional PAGE protein profiles of group I FIG. 5. One-dimensional PAGE protein profiles of different iso- spiroplasmas. Lanes: 2,4,6, 8, 10, 12, and 14, strain P40T; 1, S. citri lates of subgroup 1-8 spiroplasmas. (a) Isolates obtained in 1983. Mar0c-RgA2~(subgroup 1-1); 3, S. melliferurn BC-3T (subgroup 1-2); Lanes: 1, isolate 198; 2, isolate 263; 3, isolate 199; 4, isolate P354; 5, 5, S. kunkelii E275T (subgroup 1-3); 7, strain 277F (subgroup 1-4); 9, isolate 37; 6, isolate 341. (b) S. citri, P40T, and isolates obtained in strain LB-12 (subgroup 1-5); 11, strain M55 (subgroup 1-6); 13, strain 1984. Lanes: 1, isolate 46; 2, isolate 24; 3, isolate 36; 4, isolate 47; 5, N525 (subgroup 1-7). P40T before cloning; 6, S. citri; 7, P40T after cloning. 1'12 SAILLARD ET AL. INT. J. SYST. BACTEFUOL.

TABLE 6. Hybridization of spiroplasmal DNAs as determined by the S1 nuclease or hydroxyapatite technique Avg % hybridization with 3H-DNA of

T, of DNA ,en, % of misuaired Spiroplasma strain Subgroup s. citri Maroc-R8P qT .-

P40T 1-8 60 loob 100" 78.5 0 0 S. citri Maroc-R8A2= I- 1 lood 63 59 75.5 3 2.1 S. melliferum BC-3T 1-2 68 52 45 74.5 4 2.9 S. kunkelii E275T 1-3 57 66 63 76.5 2 1.4 277F 1-4 30 35 20 73 .O 5.5 3.9 LB-12 1-5 27 27 20 74.0 4.5 3.2 h155 1-6 26 27 ND ~~525 1-7 24 27 ND

~~~~~~~ ~

a Determined by subtracting the T, of heterologous hybrids of P40T DNA from the T,,, of the homologous hybrid of P40T DNA (78.5"C). Actual experimental hybridization percentages in two independent experiments, 74.2 and 71.1, with normalization to 100%. Actual experimental hybridization percentage in one experiment, 77.2, with normalization to 100%. Actual experimental hybridization percentages in three independent experiments, 71.0, 73.7, and 70.3, with normalization to 100%. was observed between the DNA of strain P40T and the Observzitians of plant sieve tubes by electron microscopy IINAs of S. citri Mar0c-R8A2~(subgroup 1-1) and S. kunkelii revealed large numbers of helical organisms (Fig. 6). These E275T (subgroup 1-3). About 50% hybridization was ob- periwinkles, when tested by an ELISA with specific immu- served between the DNA of strain P40T and that of S. noglobulin G against strain P40T,gave positive results (Table nwZZiferum BC-3T (subgroup 1-2). With the DNAs of repre- 7). sentative strains of subgroups 1-4, 1-5, 1-6, and 1-7, percent- ages of hybridization were smaller. The T,,, of the homolo- P40T gous DNA hybrid was 78.5"C. This value is only 1.5"C DISCUSSION lower than the T,,, of the native P40T DNA (SO.O"C), indicat- ing that in the homologous hybrid 98% of the bases are well paired. Compared with the homologous P40T hybrid, the The profile of serological cross-reactions between strain heterologous hybrids had less than 4% mispaired bases and, P40T and spiroplasmas of group I shows that the new thus, more than 96% well-paired bases, suggesting that the spiroplasma is a member of this complex. In DNA-DNA homologous sequence in the respective DNAs are well hybridization tests, strain P40T showed 59 to 66% related- con served. ness when tested against S. citri and S. kunkdii probes and Recovery of P40T proteins from graft-inoculated plants. only 52 to 45% relatedness to S. melliferum. Reactions with Periwinkles from which strain P40T had been isolated were other group I subgroups were of a significantly lower level. brought from Syria to Bordeaux, France, and kept in a In the metabolism inhibition test, strain P40T was most greenhouse. Shoots from diseased plants were grafted onto closely allied with subgroup 1-5. However, DNA-DNA hy- healthy periwinkles, and 2 months after grafting, symptoms bridization experiments as well as growth inhibition tests of yellowing could be detected on the inoculated periwinkle. indicated a more distant relationship. The G+C content of

FIG. 6. Electron micrograph of strain P40T in a sieve tube of an infected periwinkle plant. k, Micrometer. VOL. 37, 1987 SPIROPLASMA PHOENICEUM SP. NOV. 113

TABLE 7. ELISA characterization of spiroplasmas in coastal area. The G+C content of the DNA is 26 k 1 mol%. symptomatic periwinkles Type strain: strain P40T (= ATCC 43115T). Optical Antigen or sample density at ACKNOWLEDGMENTS 405 nm We express special thanks to David Rose for his efforts in the P40Tproteina(p,g/ml) performance of the biochemical studies, the filtration and choles- 40.0...... <2.00 terol tests, and the growth inhibition procedure. We also thank Ed 4.0 ...... 0.78 Clark for assistance in some serological tests, Colis Blood for 0.4 ...... 0.28 antiserum preparation, and Pierre Duthil for electrophoretic tests.

Plant extractsb LITERATURE CITED Healthy periwinkle...... 0.05 1. Aluotto, B. B., R. G. Wittler, C. 0. Williams, and J. E. Faber. P40T-infected periwinkle ...... 1.25 1970. Standardized bacteriologic techniques for the character- Graft-inoculated periwinkle‘ ...... 0.70 ization of Mycoplasma species. Int. J. Syst. Bacteriol. 20:35-58. S. citri-infected periwinkle ...... 0.04 2. Bovd, J. M. 1981. Mycoplasma infections of plants. Israel J. Phyllody-afflicted periwinkle ...... 0.06 Med. Sci. 17572-585. Aster yellows-afflicted periwinkle...... 0.05 3. BovC, J. M. 1984. Wall-less of plants. Annu. Rev. ‘ Prepared from strain P40T cultivated in SP-4 medium (48). Phytopathol. 22: 361-396. Prepared by maceration of 0.3 g of plant leaf tissue per ml. 4. BovC, J. M., C. Mouches, P. Carle-Junca, J. R. Degorce-Dumas, ‘ Healthy periwinkle graft-inoculated with a plant shoot from a periwinkle J. G. Tully, and R. F. Whitcomb. 1983. Spiroplasmas of group I. naturally infected with strain P40T. The Spiroplasma citri cluster. Yale J. Biol. Med. 56573-582. 5. Bove, J. M., C. Saillard, P. Junca, J. R. Degorce-Dumas, B. Ricard, A. Nhami, R. F. Whitcomb, D. L. Williamson, and J. G. Tully. 1982. Guanine-plus-cytosine content, hybridization per- the DNA of strain P40T was 26 k 1 mol%, a value consistent centages, and EcoRI restriction enzyme profiles of spiroplasmal with those of nearly all group I spiroplasmas (4). DNA. Rev. Infect. Dis. 4(Suppl.):S129-S136. 6. BovC, J. M., R. F. Whitcomb, and R. E. McCoy. 1983. Culture M. fascifrons leafhoppers injected with two strains of an techniques for spiroplasmas from plants. Methods Mycoplas- early uncloned passage of strain P40T were able to transmit mol. 2:225-234. the spiroplasma to aster and periwinkle plants. These plants 7. Bradbury, J. M. 1977. Rapid biochemical tests for characteriza- developed symptoms similar to those of S. citri infections of tion of the Mycoplasmatales. J. Clin. Microbiol. 5531-534. aster, and a spiroplasma indistinguishable from subgroup 1-8 8. Brinton, L. P., and W. Burgdorfer. 1976. Cellular and organisms could be cultivated from the symptomatic plants. subcellular organization of the 277F agent, a spiroplasma from These experiments demonstrate that subgroup 1-8 the rabbit tick Haemaphysalis leporispalustris (Acari: spiroplasmas are plant pathogens. It is noteworthy that the Ixodidae). Int. J. Syst. Bacteriol. 26:55A-560. Carle, C. Saillard, and J. M. BovC. three spiroplasmas pathogenic for plants, namely, S. citri 9. P., 1983. DNA extraction and purification. Methods Mycoplasrnol. 1:295-299. (citrus stubborn agent), S. kunkelii (corn stunt spiroplasma), 10. Carle, P., C. Saillard, and J. M. Bod. 1983. Determination of and subgroup 1-8 spiroplasmas, are all members of group I. guanine plus cytosine content of DNA. Methods Mycoplasmol. S. melliferum, a pathogen for but not plants (13, 16), is 1: 301-308. also a member of spiroplasma group I and is closely related 11. Chen, T. A., and C. H. Liao. 1975. Corn stunt spiroplasma: to the three plant-pathogenic spiroplasmas. isolation, cultivation and proof of pathogenicity. Science Strain P40T and the other subgroup 1-8 strains have been 188: 1015-1017. cultivated from periwinkle plants that became naturally 12. Clark, T. B. 1977. Spiroplasma sp., a new pathogen in honey- infected at various locations on the Syrian coast. However, bees. J. Invertebr. Pathol. 29:112-113. the natural vector(s) and the plant reservoir(s) of this new 13. Clark, T. B. 1978. Honey spiroplasmosis, a new problem for beekeepers. Am. Bee J. 118:18-19, 23. spiroplasma have yet to be identified. 14. Clark, T. B. 1982. Spiroplasmas: diversity of arthropod reser- Strain P40T and related spiroplasmas from periwinkles voirs and host-parasite relationships. Science 21757-59. have been shown to be members of the class Mollicutes (22, 15. Clark, T. B., R. F. Whitcomb, and J. G. Tully. 1982. 27), since they are wall-less procaryotes, are resistant to Spiroplasrnas from coleopterous insects: new ecological dimen- penicillin, and are filterable through 300-nm-pore membrane sions. Microb. Ecol. 8:401409. filters. They belong to the family Spiroplasmataceae (51,52) 16. Clark, T. B., R. F. Whitcomb, J. G. Tully, C. Mouches, C. and the genus Spiroplasma (47) because of their helical Saillard, J. M. Bove, H. Wroblewski, P. Carle, D. L. Rose, R. B. morphology, growth characteristics, requirement for sterol, Henegar, and D. L. Williamson. 1985. Spiroplasma melliferum, a new species from the honeybee (Apis mellifera). Int. J. Syst. and genomic features. We propose that these organisms be Bac teriol. 35296-308. designated Spiroplasma phoeniceum sp. nov. The taxo- 17. Clyde, W. A., Jr. 1964. Mycoplasma species identification based nomic description given below summarizes the properties of upon growth inhibition by specific antisera. J. Immunol. this organism. 92:95 8-965. Spiroplasma phoeniceum sp. nov. Spiroplasma phoeniceum 18. Cole, R. M., J. G. Tully, T. J. Popkin, and J. M. BovC. 1973. (phoe.ni’ ce.um. L. neut. adj. Phoeniceus of Phoenice, an Morphology, ultrastructure, and bacteriophage infection of the ancient country that was located on today’s Syrian coast, helical rnycoplasma-like organism (Spiroplasma citri gen. nov., referring to the geographical origin of the isolates). sp. nov.) cultured from “Stubborn” disease of citrus. J. Bacte- Chemoorganotroph. Acid produced from glucose. Hydro- rial. 115367-386. 19. Davis, R. E., I.-M. Lee, and J. F. Worley. 1981. Spiroplasma lyzes arginine. Does not hydrolyze urea. Phosphatase posi- floricola, a new species isolated from surfaces of flowers of the tive. Haemadsorbs guinea pig erythrocytes. Film-and-spot tulip tree, Liriodendron tulipifera L. Int. J. Syst. Bacteriol. positive. Cholesterol required for growth. Facultative 31:456-464. anaerobe. Temperature range, 20 to 37°C; temperature opti- 20. Degorce-Dumas, J. R., B. Ricard, and J. M. Bovk. 1983. Hybrid- mum, 28 to 32°C. Isolated from periwinkles that were ization between mycoplasma DNAs. Methods Mycoplasmol. naturally infected in various locations along the Syrian 1: 3 19-3 25. 114 SAILLARD ET AL. INT. J. SYST.BACTERIOL.

21. Eden-Green, S. J., and H. Waters. 1981. Isolation and prelimi- puff spiroplasma: a new epiphytic mycoplasma. Microb. Ecol. nary characterization of a spiroplasma from coconut palms in 8:169-180. Jamaica. J. Gen. Microbiol. 124:263-270. 41. Mouches, C., and J. M. BovC. 1983. Electrophoretic character- 22. Edward, D. G., ff., and E. A. Freundt. 1967. Proposal for ization of mycoplasma proteins. Methods Mycoplasmol. Mollicutes as name of the class established for the order 1:241-255. Mycoplasmatales. Int. J. Syst. Bacteriol. 17:267-268. 42. Mouches, C., J. M. BovC, J. Albisetti, T. B. Clark, and J. G. 23. Fabricant, J., and E. A. Freundt. 1967. Importance of extension Tully. 1982. A spiroplasma of serogroup IV causes a May- and standardization of laboratory tests for the identification and disease-like disorder of honeybees in southwestern France. classification of . Ann. N.Y. Acad. Sci. 14350-58. Microb. Ecol. 8:387-399. 24. Fos, A., J. M. BovC, J. Lallemand, C. Saillard, J. C. Vignault, Y. 43. Mouches, C., J. M. BovC, J. G. Tully, D. L. Rose, R. E. McCoy, Ali, P. Brun, and R. Vogel. 1986. La cicadelle Neoaliturus P. Carle-Junca, M. Garnier, and C. Saillard. 1983. Spiroplasma haematoceps (Mulsant and Rey) est vecteur de Spiroplasma apis, a new species from the honey-bee (Apis mellifera). Ann. citri en Mkditerranke. Ann. Microbiol. Inst. Pasteur (Park) Inst. Pasteur Microbiol. (Pans) 134A:383-397. 137A:97-107. 44. O’Hayer, K. W., G. A. Schultz, C. E. Eastman, J. Fletcher, and 25. Garnier, M., and J. M. BovC. 1983. Transfer of spiroplasmas R. M. Goodman. 1983. Transmission of Spiroplasma citri by the onto collodion membranes for electron microscopic studies. aster leafhopper Macrosteles fascifrons (Homoptera: Cicadel- Methods Mycoplasmol. 1:77-81. lidae). Ann. Appl. Biol. 102:311-318. 26. Garnier, M., M. Clerc, and J. M. BovC. 1981. Growth and 45. Razin, S., and J. G. Tully. 1970. Cholesterol requirement of division of spiroplasmas: morphology of Spiroplasma citri dur- mycoplasmas. J. Bacteriol. 102:306-310. ing growth in liquid medium. J. Bacteriol. 147:642452. 46. Razin, S., J. G. Tully, D. L. Rose, and M. F. Barile. 1983. DNA 27. Gibbons, N. E., and R. G. E. Murray. 1978. Proposals concern- cleavage patterns as indicators of genotypic heterogeneity ing the higher taxa of . Int. J. Syst. Bacteriol. 28:1-6. among strains of Acholeplasma and Mycoplasma species. J. 28. Hyman, R. W., I. Brunovskis, and W. C. Summers. 1973. DNA Gen. Microbiol. 129:1935-1944. base sequence homology between coliphages T7 and I1 and 47. Saglio, P., M. Lhospital, D. Laflkche, G. Dupont, J. M. Bod, between T3 and I1 as determined by heteroduplex mapping in J. G. Tully, and E. A. Freundt. 1973. Spiroplasma citri gen. and the electron microscope. J. Mol. Biol. 77:189-196. sp. n. : a mycoplasma-like organism associated with “Stubborn” 29. International Committee on Systematic Bacteriology Subcommit- disease of citrus. Int. J. Syst. Bacteriol. 23:191-204. tee on the Taxonomy of Moflicutes. 1979. Proposal of minimal 48. Saillard, C., and J. M. BovC. 1983. Application of ELISA to standards for descriptions of new species of the class spiroplasma detection and classification. Methods Mycoplas- Mollicutes. Int. J. Syst. Bacteriol. 29:172-180. mol. 1:471-476. 30. International Committee on Systematic Bacteriology Subcommit- 49. Saillard, C., J. Dunez, 0. Garcia-Jurado, A. Nhami, and J. M. tee on the Taxonomy of Mollicutes. 1982. Minutes of the meeting, BovC. 1978. Detection de Spiroplasma citri dans les agrumes et 27 and 30 August and 5 September 1978, Freiburg and Munich, les pervenches par la technique immunoenzymatic (ELISA). Federal Republic of Germany. Int. J. Syst. Bacteriol. C.R. Acad. Sci. 286:1245-1248. 32:261-264. 50. Saillard, C., J. C. Vignault, A. Gadeau, P. Carle, M. Garnier, A. 31. International Committee on Systematic Bacteriology Subcommit- Fos, J. M. Bove, J. G. Tully, and R. F, Whitcomb. 1984. tee on the Taxonomy of Moflicutes. 1984. Minutes of interim Discovery of a new plant-pathogenic spiroplasma. Israel J. meetings, 2 and 5 September 1980, Custer, South Dakota. Int. J. Med. Sci. 20:1013-1015. Syst. Bacteriol. 34:35&360. 51. Skripal, I. G. 1974. On improvement of taxonomy of the class 32. International Committee on Systematic Bacteriology Subcommit- Mollicutes and establishment in the order Mycoplasrnatales of tee on the Taxonomy of Mollicutes. 1984. Minutes of the interim the new family Spiroplasmataceae Fam. Nova. Mikrobiol. Zh. meeting, 30 August and 6 September 1982, Tokyo, Japan. Int. J. (Kiev) 36:462467. Syst. Bacteriol. 34:361-365. 52. Skripal’, I. G. 1983. Revival of the name Spiroplasmataceae 33. Jones, A. L., R. F. Whitcomb, D. L. Williamson, and M. E. fam. nov., nom. rev., omitted from the1980 Approved Lists of Coan. 1977. Comparative growth and primary isolation of Bacterial Names. Int. J. Syst. Bacteriol. 33:408. spiroplasmas in media based on insect tissue culture formula- 53. Stalheim, 0. H. V., A. E. Ritchie, and R. F. Whitcomb. 1978. tions. Phytopathology 67:738-746. Cultivation, serology, ultrastructure, and virus-like particles of 34. Junca, P., C. Saillard, J. G. Tully, 0. Garcia-Jurado, J. R. spiroplasma 277F. Curr. Microbiol. 1:365-370. Degorce-Dumas, C. Mouches, J. C. Vignault, R. Vogel, R. 54. Townsend, R. 1976. Arginine metabolism by Spiroplasma citri. McCoy, R. Whitcomb, D. Williamson, J. Latrille, and J. M. J. Gen. Microbiol. 94:417420. BovC. 1980. CharactCrisation de spiroplasmes isolCs d’insectes 55. Tully, J. G. 1983. Dark-field microscopy. Methods Mycoplas- et de fleurs de France continentale, de Corse et du Maroc. mol. 1:35-37. Proposition pour une classification des spiroplasmes. C.R. 56. Tully, J. G., D. L. Rose, C. E. Yunker, J. Cory, R. F. Whitcomb, Acad. Sci. Ser. D 290:1209-1212. and D. L. Williamson. 1981. Helical mycoplasmas 35. Kunkel, L. 0. 1926. Studies on aster yellows. Am. J. Bot. (spiroplasmas) from Ixodes ticks. Science 212:1043-1045. 13646-705. 57. Tully, J. G., R. F. Whitcomb, J. M. BovC, and P. Saglio. 1973. 36. Lei, J. D., H. J. Su, and T. A. Chen. 1979. Spiroplasmas isolated Plant mycoplasmas: serological relation between agents associ- from green leaf bug Trigonotylus ruficornis Geoffroy, p. 89-97. ated with citrus stubborn and corn stunt diseases. Science In H. J. Su and R. E. McCoy (ed.), Proceedings of the Republic 182:827-829. of China-United States Cooperative Science Seminars on 58. Tully, J. G., R. F. Whitcomb, H. F. Clark, andD. L. Williamson. Mycoplasma Diseases of Plants. National Science Council Sym- 1977. Pathogenic spiroplasmas: cultivation and vertebrate posium Series no. 1. National Science Council, Taipei, Taiwan. pathogenicity of a new spiroplasma. Science 195892494. 37. Manchee, R. J., and D. Taylor-Robinson. 1968. Haemadsorption 59. Tully, J. G., R. F. Whitcomb, D. L. Rose, and J. M. BovC. 1982. and haemagglutination by mycoplasmas. J. Gen. Microbiol. , a new species from the rabbit tick 50:465478. (Haemaphysalis leporispalustris). Int. J. Syst. Bacteriol. 38. Markham, P. G., R. Townsend, M. Bar-Joseph, M. J. Daniels, 32:92-100. A. Plaskitt, and B. M. Meddins. 1974. Spiroplasmas are the 60. Whitcomb, R. F., T. A. Chen, D. L. Williamson, C. Liao, J. G. causal agents of citrus little-leaf disease. Ann. Appl. Biol. Tully, J. M. BovC, C. Mouches, D. L. Rose, M. E. Coan, and 78:49-57. T. B. Clark. 1986. Spiroplasma kunkelii sp. nov.: characteriza- 39. McCoy, R. E. 1979. Mycoplasmas and yellow diseases, p. tion of the etiological agent of corn stunt disease. Int. J. Syst. 229-264. In R. F. Whitcomb and J. G. Tully (ed.), The Bacteriol. 36:170-178. mycoplasmas, vol. 3. Academic Press, Inc., New York. 61. Whitcomb, R. F., T. B. Clark, J. G. Tully, T. A. Chen, and J. M. 40. McCoy, R. E., H. G. Basham, and R. E. Davis. 1982. Powder BovC. 1983. Serological classification of spiroplasmas: current VOL. 37, 1987 SPIROPLASMA PHOENICEUM SP. NOV. 115

status. Yale J. Biol. Med. 56:453-459. N.Y. Acad. Sci. 266:26&275. 62. Whitcomb, R. F., J. G. Tully, T. B. Clark, D. L. Williamson, and 66. Williamson, D. L., and D. Poulson. 1979. Sex ratio organisms, p. J. M. Bod. 1982. Revised serological classification of 175-208. In R. F. Whitcomb and J. G. Tully (ed.), The spiroplasmas, new provisional groups, and recommendations mycoplasmas, vol. 3. Academic Press, Inc., New York. for serotyping of isolates. Curr. Microbiol. 7:291-296. 67. Williamson, D. L., J. G. Tully, and R. F. Whitcomb. 1979. 63. Whitcomb, R. F., J. G. Tully, P. McCawley, and D. L. Rose. Serological relationships of spiroplasmas as shown by combined 1982. Application of the growth inhibition test to Spiroplasma deformation and metabolism inhibition tests. Int. J. Syst. Bac- taxonomy. Int. J. Syst. Bactenol. 32:387-394. teriol. 29:345-351. 64. Whitcomb, R. F., J. G. Tully, D. L. Rose, E. B. Stephens, A. 68. Williamson, D. L., and R. F. Whitcomb. 1975: Plant Smith, R. E. McCoy, and M. F. Barile. 1982. Wall-less mycoplasmas: a cultivable spiroplasma causes corn stunt dis- prokaryotes from fall flowers in central United States and ease. Science 188:1018-1020. Maryland. Curr. Microbiol. 7:285-290. 69. Williamson, D. L., R. F. Whitcomb, and J. G. Tully. 1978. The 65. Whitcomb, R. F., and D. L. Williamson. 1975. Helical wall-free spiroplasma deformation test, a new serological method. Curr. prokaryotes in insects: multiplication and pathogenicity. Ann. Microbiol. 1:203-207.