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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. • AMV and WCMV were co-inoculated with 3:1; there was an obvious synergistic effect. • Double infection with AMV and WCMV led to an increase in both virus concentrations. • Double infection with AMV and WCMV synergistically changes host organelle ultrastructure. [ABSTRACT FROM AUTHOR]

Globally, white clover (Trifolium repens L.) is commonly infected by plant viruses. It is grown at gardens, roadsides, and public areas as ornamental plants in northern China. Some leaves present disease symptoms that are similar to those of virus infection. However, to our knowledge, no records are available from China regarding white clover (Trifolium repens L.) virus co-infection. To determine the viral species that infect white clover in China, plant samples with virus disease symptoms were collected and virion morphology and ultrastructure morphology of co-infected plants were observed by electron microscopy; viruses were detected by enzyme linked immunosorbent assay (ELISA) and reverse transcription PCR (RT-PCR). Virus co-infection was studied by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and real-time fluorescence quantitative PCR (RT-qPCR) after rub-inoculation of virus-free white clover planted in a growth chamber (25°C) with a photoperiod of 16 h, using single or several purified virions. Results showed that there were six types of symptoms, including those of shrinking mosaic, shrinking and macular mosaic, severe mottle mosaic, yellow macular mosaic, shrinkage chlorisis, and ring plaque and ring stria. The incidence rates for each symptom were 20.93, 1.48, 16.85, 59.07, 1.30, and 0.37%, respectively, based on the field investigation. Two types of viral pathogens were identified as Alfalfa mosaic virus (AMV) and White clover mosaic virus (WCMV). In mesophyll cells, virus particles with bacilliform virions formed aggregates and linear virions were bundle shaped. The detection rate of AMV was 100% in white clover samples by DAS-ELISA and RT-PCR detection, whereas that of WCMV was 83.33%. The co-infection rate was 83.33%. The relative contents of AMV and WCMV were significantly increased by 5.897- and 3.515-fold upon co-infection, when compared to that with single virus infection. We observed larger starch particles and fewer or collapsed chloroplast grana in co-infected plants; in addition, vacuoles were twisted and smaller, compared to those of healthy plants. To our knowledge, this is the first report of co-infection by AMV and WCMV in white clover, which has caused severe mosaicism and ultrastructure lesions in co-infected plants in China.