Purification of Mycoplasma-Like Organisms from Lettuce Affected with Aster Yellows Agent

Abstract

A technique was developed to partially purify the mycoplasma-like organisms (MLOs) associated with the aster yellows disease (AY). Young lettuce plants were inoculated with AY by the vector leafhopper (Macrosteles fascifrons) and kept in the greenhouse. Symptomatic diseased plants were harvested and the leaf veins were isolated and homogenized in an isolation medium consisting of mannitol, MOPs and PVP buffer, and followed by a low and high speed centrifugation. The pellet was resuspended and laid on a discontinuous density gradient which contained sucrose, percoll and MOPs. After centrifugation at 14,000 x g for 25 min, the AY-MLO fraction was collected. Removal of percoll was carried out by centrifuging the fraction at 100,000 × g for 2 h. Electron microscopy of the purified preparation showed high density of MLOs with well preserved cellular organelles identical to those observed in the phloem of diseased plants. This purification method, which permitted rapid separation of AY-MLO from plant materials under isoosmotic and low viscosity condition, may also be adapted for the purification of other plant MLOs.KeywordsDiseased PlantPathogenic BacteriumPurification MethodIsolation MediumLeaf VeinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.