Préparation des Antigenes des Mycoplasmes (MLO) Pathogènes de la Flavescence Dorée, à Partir de Tissus Végétaux

Antigen preparation from plant tissues of pathogenic mycoplasms (MLO) causing flavescence doree disease Extraction and purification of plant yellow's pathogen mycoplasms (MLO) from plant tissues is a difficult problem. It concerns indeed non cultivable, heterogeneous and fragile organisms which are localized in the fibrous and resistant phloem tissue. In a work directed by an infectivity test by leafhopper injection, our laboratory investigated the best host plant and the best extraction method for this type of pathogen. Broad bean, Vicia faba gave better extracts than Vitis vinifera. Stems are better than petioles or lamina. The best results were obtained with the top region of the stem, at the level where symptoms are apparent on young plants. The most efficient mincing method is achieved with razorblades moved alternatively by the rapid vertical movement of an electric knife. The extraction medium already published (Caudwell and Kuszala 1986) has to be modified for plants by various additional components, histidine buffer, antioxidizers (glutathion 0.2 mg per ml) and polymers 0.5 to 1 p 100 PVP or Polyclar, 1–2 p 100 PEG). 1 g of plant infected tissue is minced in 4 ml of medium. The extracts are filtered through a 100 mesh nylon cloth. After this stage the purification method goes parallel to that used for leafhopper extracts (Caudwell and Kuszala 1986). It is possible to obtain 4 × 10⁶ infectious units from 1 g of broad bean stem (for calculation, see C 1986). It is possible to obtain 4 × 10⁶ infectious units from 1 g of broad bean stem (for calculation, see Caudwell 1977). The possibility to extract MLO, either from infectious leafhoppers or from diseased plants enabled cross immunological assays involving antigens from one host and antibodies directed towards the other host antigens. The first step was the successful ISEM visualization of ttie MLOs (Caudwell et al. 1982), the second is the immunoenzymatic MLO-detection (Boudon et al. 1986).