Spiroplasma strain CN-ST (T = type strain), isolated from the gut of the cetoniine scarabaeid beetle Cotinus nitidiz, was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain CN-ST were shown by light microscopy to be helical, motile filaments. Cells in early passages exhibited strong translational motility that tended to be lost in later passages. Electron microscopy showed that the cells were bounded by a single cytoplasmic membrane with no evidence of a cell wall. The organism was not susceptible to penicillin. Strain CN-ST grew well in SM-1, MlD, and SP-4 liquid media and on solid SP-4 medium under aerobic or anaerobic conditions. The doubling time at 30"C, the optimum temperature, was 4.3 h. The strain also grew in 1% serum fraction medium. Strain CN-ST produced acid from glucose and catabolized arginine, but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was 29 f 1 mol%. The genome size was 1,770 kb (1,186 MDa). Other uncloned isolates obtained from C. nitida or the cetoniine hermit flower beetle Osmoderma eremicola exhibited similar or identical serological patterns. Since no other hosts were discovered in extensive studies, strain CN-ST (previously designated group IX) appears to represent a cluster of relatively host-specific cetoniine beetle-associated strains. Strain CN-5 (= ATCC 33827) is designated the type strain of a new species, Spiroplasma clarkii.

Two mollicutes (strains F7T [T = type strain] and F28) isolated from floral surfaces of plants growing in Morocco and France were capable of sustained growth in serum-free (or cholesterol-free) mycoplasma broth media. The two isolates were found to be genomically and serologically related. Morphologic examination of the organisms by electron and dark-field microscopic techniques showed that each strain consists of small, nonhelical, nonmotile, pleomorphic coccoid cells surrounded by a single cytoplasmic membrane. No evidence of cell walls was observed. Growth in serum-free or cholesterol-free medium was sustained only when the medium contained a Tween 80 fatty acid mixture (0.01 or 0.04%). The organisms grew rapidly in most conventional mycoplasma culture medium formulations containing horse or fetal bovine serum or a bovine serum fraction and under either aerobic or anaerobic environments. The optimum temperature for growth was 28"C, but multiplication occurred over a temperature range from 20 to 35°C. Both strains catabolized glucose and mannose, but did not hydrolyze arbutin, arginine, or urea. The molecular mass of the genome of strain F7T was determined to be about 886 megadaltons, while the base composition (guanine-plus-cytosine content) of the DNA was found to be 30.0 mol%. The two isolates were serologically unrelated to type strains of the 11 previously described Acholeplasma species and to 10 other unclassified sterol-nonrequiring mollicutes cultivated from various animal, plant, or insect sources. Strain F7 (= ATCC 49495) is the type strain of Acholeplasma seiflertii sp. nov.The occurrence of nonhelical, wall-less prokaryotes (class Mollicutes) on plant surfaces was first well documented in 1979 (9,15) when organisms with the general features of both Acholeplasma and Mycoplasma species were isolated from the surfaces of several tropical floral plants. Subsequent characterization of these strains and other new isolates from plant surfaces (22) showed that some of the strains were related to established sterol-nonrequiring Acholeplasma species that occur in vertebrate hosts (e.g., Acholeplasrna axanthum , Acholeplasma oculi, and Acholeplasma laidlawii) (9, 22). Other isolates represented either new Acholeplasma species (14, 33) or possibly sterol-requiring Mycoplasma species (9,22,26).While the presence of sterol-requiring Mycoplasma species on plant surfaces was suspected from the studies reported in 1979 to 1982 (15, 22), the possibility that these organisms might represent sterol-requiring, nonhelical forms of the genus Spiroplasrna could not be excluded at the time. The occurrence of Mycoplasma species on plant surfaces was confirmed recently when two of the strains from the early studies (17, 29) were fully characterized. In addition, sterol-requiring mollicutes were isolated from insect guts and from hemolymph samples (30), and four of these strains were characterized recently as new insect-derived Mycoplasma species (28, 34).The natural occurrence of acholeplasmas in the guts and hemolymph of in...