Double infection of Nicotiana benthamiana with AMV and WCMV increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content.

To clarify the synergistic pathogenic mechanism of Nicotiana benthamiana double infection with alfalfa mosaic virus (AMV) and white clover mosaic virus (WCMV), AMV and WCMV co-inoculation of N. benthamiana as treatment and single inoculation of AMV or WCMV and phosphate buffer solution (pH 7.0, PBS) as control, respectively. The concentrations and the relative expression of AMV and WCMV coat proteins were determined by a double antibody sandwich enzyme-linked immune sorbent assay (DAS-ELISA) and real-time fluorescence quantitative PCR (RT-qPCR) in a double infection of N. benthamiana with AMV and WCMV. Meanwhile, virion morphology, ultrastructure morphology, and chlorophyll content were observed and determined by electron microscopy. The results showed that the diseased symptoms were more serious, and virus concentration and relative expression of AMV and WCMV coat proteins were also higher in N. benthamiana double infection with AMV and WCMV than in AMV or WCMV single infection. The main symptoms manifested as severe mottle mosaic, shrinkage, and chlorosis. The concentrations of AMV and WCMV were 182.23 pg/mL and 148.77 pg/mL of double infection with AMV and WCMV, which were 1.75-fold and 1.62-fold than AMV and WCMV single infection, respectively. The relative expression of AMV and WCMV coat proteins was 4.25-fold and 2.50-fold than the single virus infection, respectively. Electron microscopy also observed that chloroplast malformation, cell membrane deformation, contents dissolution, grana lamella disorder, fat granules increased and enlarged, starch granules enlarged, and mitochondria were seriously malformed in a double infection of N. benthamiana with AMV and WCMV. The chlorophyll content was significantly lower for double infection with AMV and WCMV than for AMV or WCMV single-infected and CK, reduced by 31.52 %, 22.83 %, and 76.09 %, respectively. This is the first report of a double infection of N. benthamiana with AMV and WCMV that increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content.