Isolation and properties of filamentous viruslike particles associated with little cherry disease in Prunus avium

Abstract

Filamentous viruslike particles (VLP) associated with little cherry disease were purified directly from leaves of naturally infected field-grown and graft-inoculated, greenhouse-grown sweet cherry trees. A procedure was developed which effectively released the VLP from the phloem elements and simultaneously protected them against the denaturing and interfering agents known to become increasingly more abundant in the leaves as the season progresses. Milk was selected as the extraction medium because of its excellent protection against tannins and polyphenol oxydase and mucoid action. Milk extracts were clarified by filtration after addition of 20% (NH4)2SO4. VLP were isolated by differential centrifugation and further purified by polyethylene glycol precipitation. Electron microscopy of negatively stained preparations revealed very flexuous particles with a characteristically open, electron-transparent outer helix with pitch 4.25 nm and a prominent opaque central core. Particles had a constant width of 10.6 nm, a dominant length around 1.5 μm, and possibly a second major length category around 1.8 μm. Purified particles were very stable. They had an isoelectric point of about 4.25, degraded at 80–85 °C, fragmented at room temperature in 2% sodium dodecyl sulfate, and when denatured in urea were vulnerable to proteinase K. Levels of VLP in situ were always very low. Purified preparations from good source material contained not more than 30 μg VLP/mL.