Study of the Greening Organism (GO) with Monoclonal Antibodies: Serological Identification, Morphology, Serotypes, and Purification of the GO

A monoclonal antibody (MA) previously developed to a strain of the greening organism (GO) from Poona, India, was used to purify the homologous organism from infected periwinkles. Immunofluorescence and immunogold labeling were used to identify the GO before and after purification. Isolated cells of the GO could be observed for the first time in the electron microscope. Both filamentous forms and round forms were seen. The filamentous forms were 1 to 4 pm. The diameter of their cross section varied between 0.1 and 0.3 pm. The round forms had a diameter of 0.2 to 1 pm. We have also prepared several MA against a strain of the GO from Fujian province, China. These MA were able to detect the homologous GO strain but not the Indian strain of the GO. The MA against the Indian strain detected the homologous Indian strain as well as strains from Assam, Delhi and Shillong (India), Bali and Java (Indonesia) and Lipa city (Phillipines); they did not detect strains from China, Thailand and Malaysia; several strains from India, Indonesia and the Phillipines. These results demonstrate for the first time that different stereotypes of the GO exist, even within the same geographical area. Preparation of polyclonal antibodies and DNA probes less specific than MA is in progress to detect all GO strains. Greening disease of citrus is characterized by the presence of bacteria-like organisms (BLO) localized exclusively in the sieve tubes of infected plants (6). Morphologically speaking, the same BLO if found associated with greening disease from all geographical origins (Africa, Asia, Arabic peninsula). Even though Koch's postulates have not been fulfilled, these BLOs are considered to be the causal agent of the disease. In this paper we report the production of monoclonal antibodies (MA) against the GO from Fujian (China) and the use of such MAS as well as those prepared previously against the GO from Poona (India), for serological studies of the organism. MATERIALS AND METHODS Production of monolocal antibodies. The procedures for the production of screening of GO-specific hybridomas has been described (4). Nature of the antigens recognized by monoclonal antibodies. The nature of the GO antigens camying the epitopes recognized by the MAS was determined by immunofluorescence (IF) on midrib sections of GO-infected periwinkles previously submitted to one of the following treatments: i) heating at lOOC for 5 min; ii) digestion with proteinase K (0.5mglml) for 90 min at 37C; iii) treatment with 50mM periodate in PBS for 60 min at room temperature (RT) in the dark; iv) treatment with 1M NaCl for 30 min at RT. Each treatment was followed by extensive washing with phosphate buffer saline (PBS O.lM pH 7.0). The sections were then incubated for 30 min at RT with the various hybridoma supernatants, rinsed with PBS-tween (PBS plus 0.05% tween 20) and incubated with fluorescein isothiocyanate (F1TC)labeled goat anti mouse immunoglobulins (Diagnosis Pasteur) for 30 min at RT. After washing with PBStween, the sections were mounted in 50% glycerol in PBS pH 7.4 and observed in a Zeiss I11 RS epifluorescent microscope (filter combination BP 455-4901 FT 5101 LP 420). Electron microscopy of the GO in periwinkle extracts and in purified preparations. The collodion transfer technique (3) with or without immunogold-labeling was used for GO observation and characterization. Immunoaffinity purification of Greening, Stubborn, and Other Diseases 429 Poona-GO. Antibody 10A6 has been coupled to CNBr activated Sepharose 4B and used to purify Poona-Go by immunoaffinity. The organisms were eluted from the matrix by 0.1M glycin at pH 11.5. A detailed description of the procedure is presented elsewhere (7). Double sandwich ELISA (ds Elisa) for the detection of PoonaGO. The two hybridomas 2D12 and 10A6 were labeled with alkaline phosphatase (AP) and then used to develop a DAS-ELISA as described by Clark and Adams (1).