APPLICATION OF HYBRIDOMA TECHNIQUES IN DIAGNOSING PLANT PATHOGENIC SPIROPLASMAS AND MYCOPLASMAS (MONOCLONAL ANTIBODIES, ELISA, IMMUNOFLUORESCENT STAIN, DISEASE DETECTION, MLO)

Hybidoma techniques have been applied in the production of monoclonal antibodies for two plant pathogenic spiroplasmas and one mycoplasma-like organisms (MLO). Monoclonal antibodies against Spiroplasma citri (Maroc strain) and corn stunt spiroplasma (CSS) (strain I-747) were produced and used in the identification of spiroplasma strains. Monoclonal antibodies against the aster yellows (AY) MLO were also produced and used in the diagnosis of AY-infected plants.; Using indirect biotinylated enzyme-linked immunosorbent assay (ELISA), we found that monoclonal antibodies against S. citri (Maroc) reacted specifically with some S. citri strains but not with other strains from different geographic areas. These monoclonal antibodies also distinguished S. citri from related spiroplasmas of the same serogroup (group I) as well as 19 spiroplasma strains belonging to the 18 other serogroups. In serological specificity studies, monoclonal antibodies against CSS were used to test 32 spiroplasma straings belonging to 19 different serogroups. CSS-specific monoclonal antibodies reacted specifically with all of the three CSS strains tested, but not with any other spiroplasma strains from different groups or serogroups. However, nonspecific reactions to heterologous spiroplasma strains were obtained when conventional polyclonal antibodies (PA) against S. citri and CSS were used.; Monoclonal antibodies against the AY agent, a noncultivable MLO, were produced by using partially purified immunogen from infective insects, and diseased lettuce. In vitro and in vivo immunization techniques were applied in the hybridoma production. Seven stable hybridoma clones secreting specific monoclonal antibodies against the AY agent were obtained. These antibodies were compared with polyclonal antibodies purified from the antisera collected from immunized mice during splenectomy. Both the MA and PA were used in the in vitro and in situ detection of the AY-agent using indirect ELISA and immunofluorescent staining. MA reacted specifically to the AY-infected plants and differentiated the AY-agent from other MLOs associated with seven different yellows diseases. Both the plant antigen-absorbed and the unabsorbed PA, however, cross reacted to plant antigens and thus are unsuitable for distinctions among different MLOs. This is the first successful attempt to produce monoclonal antibodies for the noncultivable MLOs and the use of these antibodies as diagnostic tools.