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We have previously shown that Spiroplasma citri oxidoreduction sites, as revealed by the reduction of potassium tellurite into electron-dense tellurium crystals detectable by electron microscopy, were located at the blunt end of the organisms. The time of incubation of S. citri in potassium tellurite had no influence on the labeling. We have investigated the presence and location of oxidoreduction sites for other spiroplasmas such as the corn stunt spiroplasma (CSS), 277F, B88, BNR1 and PPS1. For all of these strains (except CSS, which is similar to S. citri), the location of oxidoreduction sites was affected by the time of incubation in potassium tellurite, and could be observed in different locations of the helix. 277F showed first labeling at the blunt end and a second, strong labeling at the tapered end. The other strains showed labeling all along the helix, but labeling at the blunt end generally appeared first. When Drosophila (Dm-1) cell cultures were infected with S. citri or 277F, the organisms adsorbed to the cells. Observation of infected cells by electron microscopy revealed that S. citri attached to the cells by the blunt end, while 277F attached either by the blunt end or the tapered one. The infection of leafhopper cell cultures (AS-2) with S. citri has been followed by transmission electron microscopy after incorporation of tritiated thymidine in the organism, and an immunocytochemical method has been developed to locate the organisms inside the cells.

Spiroplasma virus Group 4 was propagated, purified and characterized. The isometric capsid is made from one major protein of 60,000 Da and contains a circular single-stranded DNA molecule of 1.7 X 10(6) Da. The host-range of SV4 is limited to Subgroup I-2 spiroplasmas. The virions are released from the infected cells by a lytic process. Transfection of spiroplasmas of Subgroup I-2 by the viral DNA was demonstrated. A high efficiency of transfection was routinely obtained in the presence of polyethyleneglycol. These experiments indicate that spiroplasmas can absorb viral DNA, opening the way for cloning.

To study natural transmission of Spiroplasma citri in the Mediterranean area, periwinkles (Vinca rosea L.) were exposed to natural infection in several locations during the summer of 1983. Detection of S. citri in the periwinkles was assayed by ELISA and culture of the organisms. Some of the periwinkles developed yellows disease symptoms; they contained helical organisms in their sieve tubes, as determined by electron microscopy. A spiroplasma could be cultured from them, but their ELISA reaction for S. citri detection was negative. These results suggested that a spiroplasma different from S. citri had been discovered. The apparently new spiroplasma (P40) was examined for serological relationships with other spiroplasmas by metabolism inhibition and deformation tests. Spiroplasma P40 was found to have relatedness only to Group I spiroplasmas. Healthy periwinkles graft inoculated with shoots of the initial symptomatic periwinkle showed yellows symptoms 3 months after inoculation. Extracts of the initial and the graft-inoculated periwinkles gave positive ELISA reactions with anti-P40 IgG.

A 6-week-old female BALB/c mouse was immunized with a preparation of partially purified sieve tubes of periwinkle (Catharantus roseus) plants infected with the apple proliferation mycoplasma-like organism. After fusion of the immunized spleen cells with myeloma cells, the supernatant fluids of the resulting hybridoma cultures were screened for the presence of antibodies in a differential avidin-biotin enzyme-linked immunosorbent assay (ELISA) using sieve tube preparations from either healthy or infected periwinkles as antigens. One hybridoma clone, giving a negative ELISA with the sieve tube preparation from healthy plants and a positive ELISA with that infected ones, was selected. This hybridoma also gave a negative ELISA when tested against sieve tube preparations from periwinkles affected by citrus greening or Spiroplasma citri, but a positive ELISA when tested against phyllody-affected sieve-tube preparations.