04465nam a2200205 a 450000100080000000500110000800800410001910000190006024501760007926000230025530000090027850000200028752038600030765000120416765000180417965000120419765300140420965300250422365300110424818438402013-02-09       1978    bl uuuu     u0uu1 u    #d1 aLIMA, J. A. A.  aBlackeye cowpea mosaic virusbpurification, partial characterization, serology, and immunochemical and cytological techniques for detection of virus-infected legume seeds.  a[S.l.: s.n.]c1978  a3 p.  aResumo de Tese.  aBlackeye cowpea mosaic virus (BICMV) was increased in cowpea, Vigna unguiculata (L.) Walp., 'knuckle purple hull', and infected leaves were used for virus and cytoplasmic inclusion purification. Either n-butanol or a combination of chloroform and carbon tetrachloride was used in the clarification process. Polyacrylamide gel electrophoresis of sodium dodecyl sulfate (SDS) dissociated inclusions and virus revealed that the inclusions were made of a single protein estimated to have a molecular weight (MW) around 70.000 daltons whereas freshly purified BICMV consisted of a main protein component with a MW of 34,000 daltons and two smaller proteins with WMs of 29,000 and 27,000 daltons, Purified BICMV had a 260/280 nm absorption ration of 1.2 and a modal length of 753 nm. Freshly purified BICMV preparations showed a single sedimenting peak with s20 =157-159 s. The purified BICMV cytoplasmic inclusions had absorption spectra characteristic for proteins. Electron microscopy of purified inclusions revealed the presence of tubes showing striations with periodicities of approximately 5 nm. Antisera reactive in SDS-immunodiffusion were obtained against untreated virions, pyrrolidine degraded cell protein, and untreated BICMV cytoplasmic incluions. Reciprocal double immunodiffusion tests with SDS-treated antigens showed that BICMV is serologically unrelated to seven potyviruses and serologically related to, but distinct from: bean common mosaic virus (BCMV), bean yellow mosaic virus (BYMC), cowpea aphid-borne mosaic virus CAMV), dasheen mosaic (DMV), lettuce mosaic virus (LMV), potato virus y (PVY), soybean mosaic virus (SoyMV), tobacco etch virus (TEV), and watermelon mosaic virus-2 (WMV-2). The intragel cross-absorption technique was also used to demonstrate distinction between closely related potyviruses. Agar medium impregnated with a mixture of antisera used for serodiagnosis of BICMV and cowpea mosaic virus in cowpea. Light and electron microscopy of cytoplasmic inclusions induced by BICMV, siratro (Macroptilium atropurpureum (D.C.) Urb.) strain of BCMV (BCMV-S) and CAMV revealed that they are similar to those induced by the potyviruses from Edwardson's subdivision-l. The different reactions induced by BICMV, and CAMV in some cowpea varieties indicated that they can also be used as used as differential hosts for these three potyviruses. Sources of resistance for BICMV were found among the cowpea varieties tested. Based on its physical, biological, cytological, and immunochemical properties, BICMV can be differentiated fron any other virus that infects cowpea. Cytoplasmic inclusions induced by BICMV in cowpea and by SoyMV in soybean were detected by serology, light microscopy, and electron microscopy in hypocotyls of 4-5-day-old seedlings grown from virus-infected seeds. Immunodiffusion tests and serologically specific electrom microscopy were used to detect BICMV in hypocotyls of 4-5-day-old cowpea seedlings grown from BICMV-infected seeds. Discs of individual hypocotyls were embedded into the agar medium 4-5 mm away from the antiserum wells. Virus-specific precipitin lines formed between virus-infected hypocotyl discs and antiserum wells, whereas no reactinos were observed with healthy hypocotyls. Precipitin lines were also observed with extracts of mixtures from infected (1 g) and healthy (up to 29 g) tissues. These immunochemical techniques were also used for detecting BCMC in hypocotyls of infected 4-5-day-old Phaseolus vulgaris L. seedlings and for detecting SoyMV in infected Glycine max (L) Merr. seedlings. Single radial immunodiffusion tests with extracts or discs of cowpea hypocotyls were also useful for detecting BICMV in germinated seeds. The reliability and simplicity of the immunodiffusion tests make them suitable for use in routine seed health testing program in any laboratory.  aDoença  aPurificação  aSemente  aSerologia  aTécnica citológica  aVirose