Determination of the distribution and multiplication sites of Flavescence Dorée mycoplasma-like organisms in the host plant Vicia faba by ELISA and immunocytochemistry

Abstract

A mycoplasma-like organism (MLO) is known to be the aetiological agent of Flavescence Dorée, an important grapevine yellows disease which occurs in Europe. Indirect enzyme-linked immunosorbent assay (ELISA) was used to monitor the distribution of the pathogen in the experimental host plant Vicia faba, during the course of infection after inoculation by the leafhopper Euscelidius variegatus. Post-embedding colloidal gold indirect immunolabelling was developed to identify, without ambiguity, the various forms of MLO cells in the different infected parts of the plant, by transmission electron microscopy. Silver enhancement of the gold probe gave accurate histological and cellular localization of MLOs in tissue sections, by light microscopy. Both ELISA and immunolocalization first detected MLO in roots 17 days after inoculation with infectious leafhoppers. Small vesicles and filamentous bodies were identified as MLOs in the cytoplasm of phloem cells, and ELISA indicated the occurrence of a multiplication phase in the roots 17–24 days after inoculation. MLOs then reached the collar and systemically colonized the basal axillary shoot. They preferentially multiplied in the apical area of this growing shoot. MLO bodies were located in mature sieve tubes and in non-functional phloem cells on the periphery of the vascular bundles, and necrotic cells were observed in the phloem tissue. Electron dense and distorted MLOs were identified in these collapsed cells.