First Report of Sugarcane Mosaic Virus on Pumpkin Plants Exhibiting Mosaic and Mottling Symptoms in China

Abstract

Sugarcane mosaic virus (SCMV) is responsible for economic losses in sorghum, maize, and sugarcane worldwide. Only some monocot plants are natural hosts of SCMV. In July 2017, mosaic symptoms such as irregular chlorosis and mottling were observed in the leaves of pumpkin (Cucurbita moschata) plants at Fujian Agriculture and Forestry University, Fuzhou, Fujian Province. Seven pumpkin plants with typical mosaic symptoms were tested by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using antibodies for cucumber mosaic virus (Agdia, Elkhart, IN) and squash mosaic virus (Agdia), respectively. However, neither of these two pathogens was detected. Because the sugarcane plants growing near pumpkin plants had been confirmed to be infected by SCMV isolate FZC1 (Deng et al. 2016), we presumed that the mosaic symptoms on pumpkin plants were owing to SCMV infection. Using the SCMV-specific antibodies (Agdia), the ELISA assays showed that six of the seven pumpkin plants were infected by SCMV. To identify the strains of SCMV infecting the pumpkin plants, total RNA was extracted from leaves of all six pumpkin plants and two sugarcane plants infected by SCMV from the neighboring field using a TransZol Up Plus RNA Kit (Transgen, Beijing, China). The cDNA synthesis was performed using M4T primer (5′-GTTTTCCCAGTCACGAC[T]₁₅-3′) (Chen et al. 2001) by reverse transcription. Subsequently, polymerase chain reaction was performed using an SCMV-specific primer (SCMV7-F, 5′-ATTTATGGAGGAGTGGGAGC-3′) and M4 primer (5′-GTTTTCCCAGTCACGAC-3′) (Deng et al. 2016) with the amplification program as follows: 94°C for 4 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C 2 min, with a final extra 7 min at 72°C. The expected 1,925-bp amplicons were produced from all eight samples. The amplicons were cloned into a pMD19-T Vector System (Takara, Dalian, China) and sequenced. The sequences of the two amplicons from sugarcane plants were identical to the corresponding fragment of SCMV isolate FZC1. The sequences of the six amplicons from pumpkin plants were identical and were subjected to BLASTn search in GenBank. The results showed that the query sequence (GenBank: MK680822) had the highest nucleotide similarity, 95.97%, and the highest amino acid identity of 99.04% with SCMV isolate FZC1 (GenBank: KR108212) Phylogenetic analysis based on the SCMV coat protein showed that the pumpkin SCMV isolate was clustered into the same subgroup with FZC1. We prepared inoculum by homogenizing approximately 1 g of symptomatic leaves of SCMV-infected sugarcane plants grown in the vicinity of pumpkins in 3 ml of 0.01 M phosphate buffer (pH 7.0). Then healthy pumpkin plants (cv. BeiBei) were mechanically inoculated with the inoculum and cultured in the greenhouse under the following conditions: photoperiod 14-h light/10-h dark, light intensity 600 μmol/m²/s, temperature regime 30°C day/25°C night. Three weeks later, 7 out of 10 inoculated pumpkin plants developed mosaic symptoms, and the DAS-ELISA assays confirmed presence of SCMV in symptomatic pumpkin leaves. To our knowledge, this is the first report of SCMV infection of pumpkins and disease symptoms of this virus on a dicotyledonous plant. These findings will benefit the epidemiology and management of SCMV in China.