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Spiroplasma diabroticae sp. nov., from the Southern Corn Rootworm Beetle, Diabrotica undecimpunctata (Coleoptera: Chrysomelidae)

¹ Laboratoire de Biologie Cellulaire et Moléculaire, Institut Nationale de Recherche Agronomique, and Université Bordeaux II, 33883 Villenave d'Ornon, France
² Vegetable Laboratory, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Facility, Frederick, Maryland 21702
³ Insect Biocontrol Laboratory, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Facility, Frederick, Maryland 21702
⁴ U.S. Department of Agriculture, Beltsville, Maryland 20705; Mycoplasma Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Facility, Frederick, Maryland 21702
⁵Department of Anatomical Sciences, State University of New York, Stony Brook, New York 11794

^* Corresponding author. Mailing address: Vegetable Laboratory, GH3-1, Building 010, R2 BARCW, Beltsville, MD 20705. Phone: (301) 504-8339. Fax: (301) 504-6017.

ABSTRACT

Spiroplasma strain DU-1^T (T = type strain), which was isolated from hemolymph of the corn rootworm Diabrotica undecimpunctata (Coleoptera: Chrysomelidae), was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain DU-1^T were shown by light microscopy to be helical motile filaments. Electron microscopy revealed cells bounded by a single cytoplasmic membrane, with no evidence of a cell wall. The organism was not sensitive to 500 U of penicillin per ml. Strain DU-1^T grew well in SM-1, M1D, and SP-4 liquid media, in broth supplemented with 1% bovine serum fraction or conventional horse serum, and under both aerobic and anaerobic conditions. This organism did not appear to have a sterol requirement for growth, as has been reported for several other Spiroplasma species or strains. Optimal growth occurred at 32°C, with a doubling time of 0.9 h; strain DU-1^T multiplied at 10 to 41°C but failed to grow at 5 or 43°C. It produced acid from glucose but hydrolyzed neither arginine nor urea. The results of reciprocal serologic tests in which antigens or antisera to established Spiroplasma species, groups, subgroups, and putative groups were used indicated that strain DU-1^T was serologically distinct. This organism has a DNA guanine-plus-cytosine content of 25 ± 1 mol% and a genome size of 1,350 kbp. Strain DU-1^T is a member of a cluster of fast-growing insect-associated spiroplasmas, as determined by sequence analysis of 16S rRNA. On the basis of the results of this study and previously published data, strain DU-1 (= ATCC 43210) is designated the type strain of a new species, Spiroplasma diabroticae.