Spiroplasma citri gen. and sp. n.: a mycoplasma like organism associated with 'Stubborn' disease of citrus

Abstract

The mycoplasma like organisms observed in the sieve tubes of citrus plants affected by 'Stubborn' disease have been obtained in pure culture in various media. The cultural, biological, biochemical, serological, and biophysical properties of a California and a Morocco isolate have been determined. Classical fried egg colonies were observed. An anaerobic environment (5% CO2 in nitrogen) favored growth on solid medium. Horse serum or cholesterol was required for growth. The temperature for optimal growth was 32°C. The organisms passed through 220 nm filters. Positive reactions for glucose and mannose fermentation and phosphatase activity were obtained. Negative reactions were observed for esculin fermentation, arginine and urea hydrolysis, and serum digestion. All biochemical and biological reactions were identical for both isolates except for tetrazolium reduction and hemadsorption tests. The organisms were resistant to penicillin but sensitive to tetracycline, amphotericin B, and other inhibitors. The cell protein patterns of the 2 strains were identical to each other but clearly distinct from those for known mycoplasmas. The guanine plus cytosine content of the deoxyribonucleic acid of both strains was close to 26 mol%, and their genome size measured 109 daltons. The studies reported showed that the 2 organisms recovered from 'Stubborn' affected citrus plants comprise a single serological group and that they are serologically distinct from recognized Mycoplasma and Acholeplasma species in the order Mycoplasmatales. The cultural, biochemical, and biophysical properties of the organisms support the serological results, confirm the unique nature of these organisms, and justify their placement in a new genus, Spiroplasma, as a new species, S. citri. S. citri is the type species of the genus Spiroplasma. The Morocco strain (=R8 A2), designated as the type strain of S. citri, has been deposited in the American Type Culture Collection as ATCC 27556; the California strain (=C 189) has been deposited as ATCC 27665. The taxonomic position of S. citri is discussed. The final decision on the assignment of the citrus agent to either the class Mollicutes or the class Schizomycetes must await further analysis.