Purification of Mycoplasma-like Organisms from Lettuce with Aster Yellows Disease

Abstract

A technique was developed to partially purify the mycoplasm-like organisms (MLOs) associated with the aster yellows (AY) disease. Young lettuce plants were inoculated with the AY agent by leafhopper transmission and kept in a greenhouse. Leaf veins from symptomatic lettuce were isolated and homogenized in an insolation medium consisting of D-mannitol, 3-(N-morpholino)-propane sulfonic acid (MOPS), polyvinylpyrrolidone, L-cysteine, and ethylenediamine tetraacetic acid, before low- (8 min at 1,500 g) and high- (30 min at 35,000 g) speed centrifugation. The pellet was resuspended in suspending medium containing mannitol and MOPS and layered on a discontinuous Percoll density gradient of 15, 30, and 50% (v/v). After centrifugation at 20,000 g for 20 min, the fraction formed at the interface of the 30 and 50% solutions was collected and diluted with suspending medium. Gradient material was removed by centrifuging the diluted fraction at 100,000 g for 2 hr. Electron microscopy of thin secions made from purified preparation showed a high concentration of MLOs with well-preserved cellular structures identical to those in the phloem of disease plants. These MLO cells exhibited a trilaminar limiting membrane, cytoplasmic ribosome granules, and a network of DNA.