Species-Specific and Cross-Reactive Monoclonal Antibodies to the Plant-Pathogenic Spiroplasmas Spiroplasma citri and S. kunkelii

Jordan, R. L., Konai, M., Lee, I.-M., and Davis, R. E. 1989. Species-specific and cross-reactive monoclonal antibodies to the plant-pathogenic spiroplasmas Spiroplasma citri and S. kunkelii. Phytopathology 79:880-887. A panel of 46 murine monoclonal antibody-secreting hybridomas was with S. kunkelii (subgroup 1-3) strains. The remaining 12 monoclonal developed from mice immunized with a mixture of strains of Spiroplasma antibodies reacted with antigenic sites common in three to eight of the citri (M240H) and S. kunkelii (1-747, F32, and PU8-17). Monoclonal eight group I subgroups, including honeybee spiroplasmas, S. melferum antibodies with a range of specificities were selected in enzyme-linked (1-2), 277F spiroplasma (1-4), green leaf bug spiroplasmas (1-5), Maryland immunosorbent assay (ELISA) tests by using intact and disrupted flower spiroplasma (1-6), Cocos spiroplasmas (1-7), and the periwinkle spiroplasma cells and purified spiroplasma cell membrane preparations. spiroplasma, S. phoeniceum (1-8). None of the monoclonal antibodies Forty of the monoclonal antibodies recognized antigenic sites found on reacted with any non-group I spiroplasmas tested. At least 17 different Ig-trapped intact spiroplasmas and spiroplasma membrane preparations, antigenic sites were defined by the 46 monoclonal antibodies. These whereas the remaining six monoclonal antibodies reacted only with discriminatory serological reagents will be useful for the detection and disrupted spiroplasma cells in indirect ELISA. When used with 36 strains identification of spiroplasmas in plants and insects and for providing of spiroplasmas representing group I and groups III-XI spiroplasmas, information on the antigenic relationships among other spiroplasma 17 monoclonal antibodies reacted specifically with strains of S. citri strains and newly recognized spiroplasmas. (subgroup 1-1), and an additional 17 monoclonal antibodies reacted only Additional keywords: citrus stubborn, corn stunt, differentiation, identification, serologic relationships, serology, spiroplasma serogroups. Spiroplasmas are a group of motile, helical, wall-free of hybridoma technology (21). The advent of monoclonal antibody prokaryotes. Several spiroplasma species are plant or insect production has provided homogenous and biochemically defined pathogens. They include Spiroplasma citri, the causal agent for immunological reagents with unique specificities and extreme citrus stubborn disease (31) and horseradish brittleroot disease discriminatory abilities. Monoclonal antibodies have an (14); S. kunkelii, the etiological agent of corn stunt disease (33,39); advantage over polyclonal antisera in serological tests because S. phoeniceum, the causal agent of a yellowing disease of they react with a single antigenic determinant (epitope) rather periwinkle (32); and S. melliferum, which is lethal to the honeybee than with many different epitopes and/ or antigens. In addition, (7). Spiroplasmas have also been isolated from other insects monoclonal antibodies specific to unique epitopes can be (5,10,26) and from the surfaces of flowers of several plant species generated even when nontarget antigens or "mixed" immunogens (10,13,36). are used for immunization, as long as a target-specific screening Twenty-three spiroplasma groups and eight subgroups have assay is used to select the desired monoclonal antibodies (17,18). been proposed, based on serological, biochemical, genomic, and M onoclonal antibodies specific for various plant pathogens, cultural comparisons (38). Group I, referred to as the S. citri including viruses, bacteria, and mycoplasmalike organisms, have complex (3), contains eight subgroups with complex patterns of been produced and used in diagnostic and taxonomic studies serological and genomic interrelationships. These spiroplasmas (15,17). show various degrees of heterologous reactions in several Two separate panels of monoclonal antibodies produced by serological tests, including growth inhibition, cell deformation, Lin and Chen to S. citri (27) and S. kunkeiji (28) were speciesmetabolic inhibition, and ELISA (2,7,10,31-35). DNA-DNA specific and did not cross-react with any other spiroplasmas tested. hybridization, EcoRI restriction mapping of spiroplasma DNAs, All of the S. citri-specific monoclonal antibodies reacted with and spiroplasma protein patterns support the serological results all of the nine U.S. strains and one North African strain tested (4,7,23,31-33). Groups II through XXII are serologically distinct but did not react with four strains isolated in Algeria, Iran, and from group I and from each other (3,4,9,34,35,38). Israel (27). Serology has been a most useful tool in the classification, The aims of the work presented here were to produce S. citriidentification, and detection of spiroplasmas. The specificity of specific, S. kunkelii-specific, and cross-reactive monoclonal serological techniques has been enhanced with the introduction antibodies to be able to identify and differentiate strains of S. citri and S. kunkeiji and to differentiate both S. citri and S. kunkeiji from other serovar subgroups and species of Spiroplasma, This article is in the public domain and not copyrightable. It may be freely adt netgt h eooi eainhp mn prpam reprinted with customary crediting of the source. The American strains within group I. We report here the isolation and Phytopathological Society, 1989. characterization of species-specific and group-specific monoclonal