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; Mycoplasma 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 spiroplasma 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 spiroplasmas 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 Taxonomy 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 leafhoppers (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 Spiroplasma citri (47), the etiological agent of citrus 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 Mollicutes (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 Drosophila 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