3.3.24
PRODUCTION, CHARACTERIZATION AND APPLICATION OF MONOCLONAL ANTIBODIES AGAINST XANTHOMONAS CAMPESTRIS PV. MANGIFERAE-INDICAE, X. C. PV. VESICATORIA AND RALSTONIA SOLANACEARUM

K TSUCHIHA, C D'URSEL, F IFTIKHAR, M HORITA and Y NOZU

National Institute of Agrobiological Resources (NIAR), 2-1-1, Kannondai, Tsukuba,305-0856, Japan

Background and objectives
Bacterial spot diseases of mango, pepper and tomato, and bacterial wilt of solanaceous plants caused by Xanthomonas campestris pv. mangiferae-indicae (XCM), X. c. pv. vesicatoria (XCV) and Ralstonia solanacearum (RS), respectively, are major constraints of yield losses in tropical and in some warm temperate regions of the world. Production of the monoclonal antibodies (MABs) that could give taxonomic information for specific detection of the pathogens in epidemiological studies of the diseases was conducted.

Materials and methods
Bacterial strains tested were collected from worldwide. ABC-ELISA, one of the indirect methods, was adopted routinely for screening and characterization of MABs as well as detection of bacteria from the infected plants. Both immunoblotting and immunogold labelling were used for antigen analysis related to the MABs.

Results and conclusions
Fourteeen and 11 MABs were produced against a white strain of XCM and an atypical yellow one, respectively. Among all the MABs and all the isotypes found, the majority were IgM. The dilution limit was between 100 and 20,000, and was 10 times higher with the corresponding ascitic fluid. Their reaction pattern could relate white and yellow XCM and Japanese with worldwide XCM. Two MABs, 1A7H12G3 and 1E2E1, could be selected for practical use because of their high specificity and reactivity. These two MABs bound to typical LPS bands between 41 and 133 kDa in immunoblotting.

Three representative MABs, 7AH10, 5HB3 and 4AD2, were selected from a total of 90 hybridomas raised against XCV strains UPB141, MAFF 301294 and 2625. All of them were IgM. The dilution limit of 7AH10 and 5HB3 was 125,000, and that of 4AD2 was 24,000. For all the MABs the detection limit was 10 times lower when a competitive ELISA format was used, and then comprised between 104 and 103 c.f.u./ml. With the 32 XCV strains isolated from Japan, Guadeloupe, Maroc, Senegal, Reunion or France, 30 strains (94%) reacted with at least two of three MABs. Particularly, the 17 from Japan were all recognized, and strains of tomato and pepper reacted specificaly with 4AD2 and 7AH10 , respectively. The MAB 7AH10, obtained against the UPB141, an Amy- strain reacted with all the Amy- (n=20) and 15% (n=19) of the Amy+ strains. And against the 2625 strain, an Amy- strain, the MAB 5HB3 recognized 94% of all the XCV (n=39). For all the MABs, cross-reactions with other pathovars or species were less than 4% (n=67). In immuno-blotting, the three MABs reacted with typical LPS bands between 41 and 120 kDa from EDTA extract and water phase after phenol extraction. Thirteen MABs were established from an immunization of live cells of an RS strain MAFF 301520 (race 1, biovar 4) . They were all IgM. They were divided into groups based on reactivity to the strains from tobacco, potato, ginger and eggplant.

References
1. Tsuchiya K, Takahashi Y, Shohara K et al., 1991. Annals of the Phytopathological Society of Japan 57, 196-202.
2. Tsuchiya K, d'Ursel C, Nozu Y, 1995. European Journal of Plant Pathology 645, Abstracts.