Metaplexis japonica (Thunb.) Makino, a species of family Apocynaceae, is well known as a traditional herbal medicine and has a wide distribution in China. In September 2016, leaves of M. japonica plants growing near a pumpkin field in Zhenjiang, Jiangsu province, exhibited light and dark green mosaic and deformation symptoms characteristic of virus infection. Crude sap of symptomatic M. japonica leaves was prepared by adding 500 μl of extraction buffer (0.01 M phosphate buffered saline [pH 7.4], 1% wt/vol Na2SO3, and 0.05% vol/vol Tween-20) to 50 mg of leaf tissue ground in liquid nitrogen. Mechanical inoculation with the sap was performed on the young, fully expanded leaves of the 6-leaf stage Nicotiana... More
Metaplexis japonica (Thunb.) Makino, a species of family Apocynaceae, is well known as a traditional herbal medicine and has a wide distribution in China. In September 2016, leaves of M. japonica plants growing near a pumpkin field in Zhenjiang, Jiangsu province, exhibited light and dark green mosaic and deformation symptoms characteristic of virus infection. Crude sap of symptomatic M. japonica leaves was prepared by adding 500 μl of extraction buffer (0.01 M phosphate buffered saline [pH 7.4], 1% wt/vol Na2SO3, and 0.05% vol/vol Tween-20) to 50 mg of leaf tissue ground in liquid nitrogen. Mechanical inoculation with the sap was performed on the young, fully expanded leaves of the 6-leaf stage Nicotiana benthamiana plants, resulting in leaf distortion at 5 days postinoculation (dpi) and mosaic and plant stunting symptoms at 30 dpi. Mock-inoculated N. benthamiana plants did not exhibit any symptoms at 5 or 30 dpi. Double-stranded (ds) RNAs extracted from the symptomatic leaves using a previously described method (Morris et al. 1979) were subjected to 6% polyacrylamide gel electrophoresis. After silver staining, four dsRNA approximately 4.0, 3.5, 2.7, and 1.7 kb in size were observed. These results suggested that the M. japonica plants with mosaic and deformation symptoms were most likely infected by an RNA virus. To better understand the genomic information of this virus, dsRNA extracted from the symptomatic leaves of M. japonica plants served as a template for sequence-independent amplification (SIA) (Agindotan et al. 2010; Bohlander et al. 1992). Polymerase chain reaction (PCR) products were ligated into pUCm-T Vector (Sangon Biotech, Shanghai) and sequenced (GenScript, Nanjing, China). BLAST analysis showed that a 630-nt sequence shared the highest (98%) nucleotide identity with a Cucumber mosaic virus (CMV) isolate from China (KC218822), and a 1,151-nt sequence shared the highest (96%) nucleotide identity with a CMV subgroup IB isolate from China (FJ268745), suggesting CMV was the causal agent. This isolate was tentatively named CMV-Met. Reverse transcription (RT) PCR detection was performed using the CMV-specific primer (forward, 5′-TAACTTTAGAGTCCTGTCGC-3′; and reverse, 5′-ATGAAGTACTAGCTCATCCG-3′) designed using the obtained coat protein (CP) nucleotide sequence. The expected RT-PCR product was amplified from symptomatic N. benthamiana plants inoculated with sap but was not amplified from the asymptomatic N. benthamiana plants or negative controls. Subsequently, the complete sequence of RNA3 of CMV-Met (GenBank KY794710) was determined by sequencing RT-PCR products obtained using degenerate CMV primers and CMV-Met-specific primers designed from sequences obtained by SIA. Phylogenetic analysis based on the CP amino acid sequence and the RNA3 5′-nontranslated region of CMV-Met and other CMV strains using MEGA 5.1 software indicated that CMV-Met was clustered in subgroup IB (Roossinck et al. 1999). This finding is helpful in developing strategies for controlling disease caused by CMV in agricultural areas growing M. japonica. To our knowledge, this is the first report of infection in M. japonica by CMV.
