Detection of Bean Golden Mosaic Virus in Host Tissues and Infectivity of Its Genome in Bean Mesophyll Protoplasts

145 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1983.Indirect fluorescent antibody (F-Ab) staining, enzyme-linked immunosorbent assay (ELISA), and DNA-DNA hybridization on nitrocellulose (dot blot) were used to detect BGMV in P. vulgaris (Top Crop). F-Ab staining of sections from leaves, stems, and roots showed that viral antigen was present in a limited number of mesophyll cells, in some sieve elements and phloem parenchyma of trifoliolate leaves, and in sieve elements and phloem parenchyma of the stem. No viral antigen was detected from the roots. Isolated cells from trifoliolate leaves showed BGMV-specific fluorescence in the nucleus and cytoplasm. By ELISA, virus titers in trifoliolate leaves reached a maximum of 73 to 125 (mu)g per gram tissue between 6 to 18 days after inoculation (DAI) of the primary leaves. Maximum titers in stems were 1.0 to 1.8 (mu)g per gram tissue between 16 to 22 DAI, and in inoculated primary leaves 0.10 to 0.15 (mu)g virus per gram between 18 to 26 DAI. Dot blots of sap or nucleic acid extracts from trifoliolate leaves showed signals even well into the chronic infection stage when viral titer by ELISA was low. These results suggest that BGMV genome exists in some stable form other than as an intact virion in chronically diseased plants.High yields of viable protoplasts were obtained from the primary leaves of Top Crop beans treated for 4 hours in a mixture of Cellulase, Macerozyme, mannitol, and 2-N-morpholinoethane sulfonic acid (MES). Protoplast yield and viability were affected by the age of the source plants, enzyme concentration, pH, and the osmotic conditions. The protoplasts were infected with bean golden mosaic virus (BGMV) viral (single-stranded) DNA. F-Ab staining showed that up to 45% of the protoplasts were infected. Nucleic acid sequences complementary to the viral genome were detected by DNA-DNA hybridization after inoculation of the protoplasts. These findings confirm that bean mesophyll cells are susceptible to BGMV infection.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD