Immunosorbent electron microscopy and gold label antibody decoration of MLOs from crude preparations of infected plants and vector insects

Abstract

A simple method is given for immunotrapping, gold labelling and electron microscopy of mycoplasmalike organisms (MLOs) from crude preparations of infected plants or insects, enabling MLOs to be confidently distinguished from host materials in negative stain preparations. The appearance of the MLOs is described. MLOs were trapped from extracts of infected periwinkle plants (Catharanthus roseus) and infected vector leaf hoppers (Macrosteles quadripunctulatus), using electron microscope grids coated with the F(ab)2 portion of specific rabbit IgGs. The MLOs were then decorated with the intact igG and labelled with goat anti‐rabbit IgG conjugated to gold particles 5 or 15 nm in diameter. Preparations were negatively stained in 0.5% ammonium molybdate or left unstained. The MLOs used were European aster yellows (EAY) from plant and vector, and Australian tomato big bud (TBB) from the plant. Preparation time was 4 h; large numbers of MLOs and MLO fragments could be trapped and identified, and EAY could be confidently distinguished from the serologically unrelated TBB. Morphologically, EAY and TBB were indistinguishable, both presenting spheroidal bodies, tubular forms and apparently budding chains of cocci. Bacilliform virus‐like particles were sometimes associated with EAY MLOs, especially those from the vector.