Location: National Germplasm Resources LaboratoryTitle: First report of pea enation mosaic virus 1 and pea enation mosaic virus 2 from Pea in China
|CHEN, XIAOJIAO - Yunnan Agricultural University|
|LI, KENUA - Yunnan Agricultural University|
|LUO, HENMING - Yunnan Agricultural University|
|HAN, SHU - Yunnan Agricultural University|
|TAN, GUANLIN - Yunnan Agricultural University|
|LI, FAN - Yunnan Agricultural University|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2020
Publication Date: 8/27/2020
Citation: Chen, X., Li, K., Luo, H., Han, S., Tan, G., Li, R., Li, F. 2020. First report of pea enation mosaic virus 1 and pea enation mosaic virus 2 from Pea in China. Plant Disease. https://doi.org/10.1094/PDIS-05-20-1123-PDN.
Interpretive Summary: Pea (Pisum sativum L.) is an economically important legume crop worldwide. It is consumed in fresh and dried form, and the pods and shoots are eaten in some cultures. The pea plant is susceptible to many aphid-transmitted viruses that can produce diseases individually or in combination. Pea enation mosaic is a major disease caused by a mixed infection of two viruses, pea enation mosaic virus 1 and pea enation mosaic virus 2 (PEMV-1 and PEMV-2). In 2019, pea plants with enation mosaic symptoms were observed in pea fields in the Yunnan Province of China. In this study, seven known viruses, including PEMV-1 and PEMV-2, were identified from those diseased plants using advanced sequencing technology. The viruses were transferred to pea seedlings, which subsequently developed symptoms. This is the first report of PEMV-1 and PEMV-2 in China, and the genome information and detection protocols developed in this study can be used to further study pea viral diseases.
Technical Abstract: Pea enation mosaic is an important virus disease of pea (Pisum sativum L.) caused by two virus species in an obligate symbiosis, Pea enation mosaic virus 1 (PEMV-1, Enamovirus, Luteoviridae) and Pea enation mosaic virus 2 (PEMV-2, Umbravirus, Tombusviridae) (citation). In November 2019,foliar yellow mosaic and vein enations symptoms that were similar to those caused by these two viruses were observed from pea plants in fields of Honghe autonomous prefecture, Yunnan province, China. The disease incidences were 20-40% in some fields. Leaves with typical virus-like symptoms were collected from five symptomatic pea plants and used for total RNA extraction. The five extracts of equimolar quantities were pooled into a sample and subjected to High Throughput Sequencing (HTS) by Illumina HiSeq system. Analyses of raw RNA reads were performed using CLC Genomics Workbench 12 (Qiagen). A total of 60,009,746 RNA reads were obtained from the sample, and de novo assembly of the reads using the CLC Genomics generated 88,105 contigs. BLAST searches of the contigs revealed the presence of contigs with high similarities to PEMV-1, PEMV-2, broad bean wilt virus 2, cucumber mosaic virus, pea seed-borne mosaic virus, bean yellow mosaic virus and Brassica yellows virus. To confirm the presence of PEMV-1 and PEMV-2 in the individual samples, two virus-specific primer pairs were designed based on the contig sequences obtained by HTS in this study. Primer pairs PEMV-1F/PEMV-1R (5’-ATGCCGACTAGATCGAAATC-3’/5’-TCAGAGGGAGGCATTCATTA-3’) that flank the cp gene of PEMV-1 and PEMV-2F/PEMV-2R (5’-ATGACGATAATCATTAATG-3’/5’-TCACCCGTAGTGAGAGGCA-3’) that target the ORF3 region of PEMV-2 were used to amplify the two viruses in reverse transcription-polymerase chain reaction (RT-PCR), respectively. Amplicons of the expected DNA fragments of 570 bp (PEMV-1) and 693 bp (PEMV-2) were obtained from all five samples, respectively. The RT-PCR products were cloned and sequenced. Seuqence analyses showed that the 570-bp amplicon (GenBank Accession No. MT481989) shared the highest nucleotide sequence identity of 98.95% with PEMV-1 (GenBank Accession No. Z48507), while the 693-bp fragment (MT481990) had the highest nucleotide sequence identity of 97.4% with PEMV-2 isolate JKI (MK948534). One gram of the symptomatic leaves from each of the five plants was homogenized with 5 mL of 0.01 M phosphate-buffered saline, pH 7.0. Each of the resulted saps was used to inoculate x healthy pea seedlings. Inoculated plants developed yellowing and mottling 10 days post inoculation (dpi), but no symptoms were observed on control plants inoculated only with phosphate-buffered saline. The enation formation was observed along the veins of the lower side of the symptomatic leaves of the inoculated plants at 30 dpi. To our knowledge, this is the first report of PEMV-1 and PEMV-2 in China. Infections of the two viruses can cause severe yield losses, and therefore, integration of detection and control measures is important in pea production in regions where the two viruses are found.