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Purification of Mycoplasma-like Organisms from Lettuce with Aster Yellows Disease. Y. P. Jiang, Graduate student, Department of Plant Pathology, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903; T. A. Chen, Professor, Department of Plant Pathology, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903. Phytopathology 77:949-953. Accepted for publication 17 December 1986. Copyright 1987 The American Phytopathological Society. DOI: 10.1094/Phyto-77-949.

A technique was developed to partially purify the mycoplasma-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 isolation 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 sections made from purified preparation showed a high concentration of MLOs with well-preserved cellular structures identical to those in the phloem of diseased plants. These MLO cells exhibited a trilaminar limiting membrane, cytoplasmic ribosome granules, and a network of DNA.