Abstract: 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.

Key words:  clover ,  AMV ,  WCMV ,  viral co-infection ,  virus-induced pathological changes ,  DAS-ELISA ,  RT-PCR ,  RT-qPCR