Showing papers in "Phytopathology in 1976"

Assay for viruses and mycoplasmas using serologically specific electron microscopy.

Abstract: DERRICK, K. S., and R. H. BRLANSKY. 1976. Assay for viruses and mycoplasmas using serologically specific electron microscopy. Phytopathology 66: 815-820 Serologically specific electron microscopy (SSEM) was debris on SSEM grids. The dilution of antiserum used to further developed as a diagnostic technique for plant viruses, prepare SSEM grids did not have a significant effect on the The technique was shown to apply to a variety of plant number of virus particles attached, except at extremely high viruses and to the corn stunt mycoplasma (CSM). Assays of antiserum dilutions. In assays of young cultures of CSM and barley yellow dwarf virus in crude extracts were easily done CSM-infected corn, filamentous forms were specifically using a mouse antiserum produced with a partially purified attached to SSEM grids; in assays of older cultures only virus preparation. The addition of sucrose (to 0.4 M) to virus spherical forms, 0.1-0.2 /m in diameter, were observed. extracts and washing buffers greatly reduced the amount of Additionalkey words: maize mosaic virus, tomato spotted wilt virus, maize chlorotic dwarf virus, maize dwarf mosaic virus. We have given reports (2, 5, 6) on a method, which has Agricultural Research Service, Beltsville, Maryland. been named serologically specific electron microscopy Barley yellow dwarf virus (BYDV) in 'Barsoy' barley was (SSEM), that provides for specific attachment of virus provided by M. A. Newman, West Tennessee Experiment particles to electron microscope grids. Grids with Station. Mouse antiserum to BYDV was produced using Parlodion films are coated with an adsorbed film of viral partially purified virus prepared by the method of antiserum. Virus particles are attached and concentrated Brakke and Rochow (1) through the first cycle of on the surface of the grids floated on extracts of virusdifferential centrifugation. Three white mice were given a infected tissue. Salts and other components in the extract total of four intraperitoneal injections of the virus are removed by washing, and the virus particles are preparation, suspended in Freund's incomplete adjuvant, stained for examination with the electron microscope. In at weekly intervals. Terminal bleedings were made 14 addition to being a very sensitive method for detection days after the last injection. Each mouse was injected with and identification of viruses, SSEM provides for a total of I ml of the virus preparation, which represented quantitative assay of viruses in crude extract (5) and has the yield from 15 g of infected barley. A control serum was been used to monitor aggregation and degradation of prepared using a similar preparation from healthy barley. virus particles that may occur during purification Of the three antisera, the one used in this report was procedures (6). The technique was used by Shalla et al. (9) superior, one gave considerably less virus attachment, to study viruslike particles in chloroplasts of plants and one failed to give detectable virus attachment. Since it infected with tobacco mosaic virus. Additional details on was not necessary to know accurately the dilution of a the use of SSEM as a diagnostic method are given in this serum (provided it was less than 1:10,000) to use it in report. SSEM, trial bleedings from mice were made by tail clipping. The tails were clipped and immediately placed in MATERIALS AND METHODS a drop of Tris-NaCl; blood cells were allowed to coagulate and settle, and SSEM grids were prepared from Viruses and antisera.--Potato virus Y (PVY) and the resulting drop of dilute serum. Terminal bleedings, by tomato spotted wilt virus (TSWV) and antisera to PVY opening chest cavities, gave approximately one-half ml of and TSWV (prepared by G. V. Gooding, North Carolina serum, which is an adequate supply for SSEM assays. State University) were provided by L. L. Black, Louisiana Procedure for serologically specific electron State University. Maize chlorotic dwarf virus (MCDV) microscopy.-Cohen-Pelco handle grids, 74 Am (200and antiserum were supplied by D. T. Gordon, Ohio mesh), were dipped in a 1% solution of polybutene in Agricultural Research and Development Center. Maize xylene and allowed to dry. Films were put on the grids mosaic virus (MMV) was collected in Louisiana by M. J. using a 0.5% solution of Parlodion in amyl acetate. The Giamalva, Louisiana State University. Antiserum to grids were carbon-coated in a vacuum evaporator. The MMV (prepared by F. Herold) was provided by R. grids were floated on drops of antiserum diluted 1:1,000 Lastra, Instituto Venezolano Investigaciones.Cientificas, to 1:10,000 with Tris buffer (0.05 M, pH 7.2) for 30 Caracas, Venezuela. Antiserum to maize dwarf mosaic minutes. Unadsorbed serum proteins were removed by virus (MDMV-A) was ATCC AS 58. Corn stunt floating the grids (five times) on drops of Tris buffer or by mycoplasma (3, 10) in culture, in corn plants, and placing the grids in a stream of buffer from a wash bottle. antiserum to it were gifts from R. F. Whitcomb, Prepared SSEM grids have been washed with distilled