First Report of Cowpea Mild Mottle Virus Infecting Soybean in China

Abstract

Cowpea mild mottle virus (CPMMV) is a positive-strand RNA virus of the genus Carlavirus in the family Betaflexiviridae that is transmitted by the whitefly Bemisia tabaci in a nonpersistent manner. It was first reported to infect cowpea in Ghana (Brunt and Kenten 1973) and subsequently found in Fabaceae and other crops across the world (Laguna et al. 2006; Zanardo et al. 2014). CPMMV causes mosaic, dwarfism, and stem necrosis in soybean (Glycine max L. Merr.), one of the most important oil crops in the world. There are no confirmed reports or information about CPMMV on soybean in China. In September 2019, leaf mosaic and crinkling symptoms were observed on soybean (cv. Zhonghuang 33) during field surveys at three locations in Anhui Province, China. The disease incidence was estimated as 20 to 40% across the field. Leaves with typical virus-like symptoms were collected from five symptomatic plants and analyzed by high-throughput sequencing (HTS) to investigate the etiology. Total RNA was extracted by TRIzol Reagent (Invitrogen, U.S.A.), and approximately 10 μg of total RNA was used for transcriptome sequencing. The final cDNA library was constructed using the TruSeq Stranded mRNA Library Prep Kit (Illumina, U.S.A.) and sequenced on an Illumina HiSeq 4000 (LC Sciences, U.S.A.). The average insert size for the paired-end libraries was 300 bp (±50 bp). A total of 30,098,814 raw reads were obtained, and the reads were assembled using Trinity software (version 2.8.5). The assembled contigs were searched against NCBI virus RefSeqs (ftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral) using a BlastX algorithm with a cutoff E value of ≤10⁻⁵. Thirty-two contigs were similar to CPMMV, which was the only plant virus (E value ≤10⁻⁵; contigs ≥200 nt) found in our HTS data. The largest contig of 8,256 bp shared 95.7% nucleotide identity to a CPMMV isolate (accession no. KY420906) sequenced from cowpea in Hainan, China. Phylogenetic analysis showed the isolate from China clustered separately from isolates from other countries. The sequence of the CPMMV isolate from Anhui was submitted to NCBI under the accession number MN908944. Mechanical transmission assays were performed using sap from symptomatic field plants and two soybean varieties (Williams 82 and Kefeng 1, 10 plants per variety). Eight of the Williams 82 plants and all Kefeng 1 plants showed systemic mosaic 14 days postinoculation, whereas control plants inoculated with phosphate-buffered saline (three per cultivar) were all symptomless. To confirm the transmission, an RT-PCR assay was performed on mechanically inoculated plants using primers designed to be specific for the CPMMV-coat protein (5′-CTGGCCAAGTGGTTTGTTTT-3′; 5′-AACCGGGTTTACAATCCACA-3′). The expected 1,865-bp PCR products were obtained from each of the five symptomatic plants tested but not from symptomless control plants. The PCR products were directly sequenced and were almost identical (99.8% nt) to the corresponding HTS-derived sequences of the isolate from Anhui province (MT232837). To the best of our knowledge, this is the first report of soybean infected by CPMMV in China. These findings show that CPMMV is a potentially important soybean disease in China that has implications for disease control and resistance breeding. This study is therefore an alert for local growers and those involved in disease management and will provide impetus and information for breeders in soybean disease resistance.