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The complete genomic sequence of a novel potyvirus from Paris yunnanensis was determined by high-throughput sequencing then confirmed by Sanger sequencing. Its genomic RNA consists 9600 nucleotides (nt) excluding the 3’-terminal poly (A) tail, containing a typical large open reading frame (ORF) of potyviruses and encoding a putative polyprotein of 3098 amino acids (aa). Pairwise comparison analysis showed the virus shares sequence identity with other members of Potyvirus was 53.0% to 57.8% at genome sequence level, and 39.3% to 51.2% at polyprotein sequence level. Phylogenetic analysis indicated that the virus was most closely related to the subgroup of plum pox virus and that of chilli veinal mottle virus within the genus Potyvirus. These results suggest that the virus should be considered as a distinct species within the genus Potyvirus and was tentatively named as “Paris mottle associated virus” (PMaV).

Pea (Pisum sativum L.) is an economically important legume crop that is commonly used as dry beans, fresh peas, pods and shoots (Guo et al. 2009). Pea enation mosaic is an important virus disease of pea caused by two viruses in an obligate symbiosis, pea enation mosaic virus 1 (PEMV-1, Enamovirus, Luteoviridae) and pea enation mosaic virus 2 (PEMV-2, Umbravirus, Tombusviridae) (Hema et al. 2014). In November 2019, foliar yellow mosaic and vein enations symptoms were observed from pea plants in five fields of Honghe autonomous prefecture, Yunnan province, China. Incidence of symptomatic plants ranged from 20 to 40% and was distributed in both small and large fields. Leaves with typical virus-like symptoms were collected from five symptomatic pea plants in two fields 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. BLASTN searches revealed the presence of contigs with high similarities to PEMV-1, PEMV-2, Pea seed-borne mosaic virus, and Bean yellow mosaic virus. To confirm the presence of PEMV-1 and PEMV-2 in the 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 RT-PCR. DNA fragments of the expected sizes (PEMV-1, 570 bp; PEMV-2, 693 bp) were amplified from all five samples. The RT-PCR products were cloned and sequenced. Sequence analysis showed that the 570-bp amplicon (MT481989) shared the highest nucleotide sequence identity of 98.95% with PEMV-1 (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 (PBS buffer), pH 7.0. Each of the resulted saps was used to inoculate onto five healthy pea seedlings. A total of 25 healthy pea seedlings were inoculated, and 16 inoculated plants developed yellowing and mottling at 10 days post inoculation (dpi); no symptoms were observed on control plants inoculated only with PBS buffer. The formation of the typical enation was observed along the veins of lower side of the symptomatic leaves of the inoculated plants at 30 dpi. PEMV-1 and PEMV-2 infection were confirmed by RT-PCR assays using the specific primer pairs described above. Although the presence of the pea enation mosaic virus complex was suspected in China based on symptomatology (Brunt et al. 1997), to our knowledge, this is the first molecular confirmation of PEMV-1 and PEMV-2 occurrence in China. The co-infection of PEMV-1 and PEMV-2 usually cause severe yield losses; therefore, integration of detection and control measures is important in pea production regions where the two viruses occurred.

Tomato mottle mosaic virus, a new species in the genus Tobamovirus and family Virgaviridae, was first reported on tomato in Mexico in 2013 (Li et al. 2013). Subsequently, tomato mottle mosaic virus (ToMMV) was found infecting pepper and eggplant in China (Li et al. 2014; Chai et al. 2018). ToMMV was thought to pose a serious risk to solanaceous crops due to its potential to break resistance and numerous transmission ways (Li et al. 2020), meanwhile, some plant species in the families of Amaranthaceae, Brassicaceae, Cucurbitaceae, and Verbenaceae, were found as the hosts of ToMMV under experimental condition (Sui et al. 2017; Li et al. 2020). To clarify the occurrence of ToMMV in Yunnan province of China, 395 plant samples, including tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.) and pea (Pisum sativum L.), exhibiting viral-like symptoms were collected in major crop plantations in Yunnan province in 2020. Total nucleic acids were extracted from the diseased samples using a CTAB method (Li et al. 2008), and tested by reverse transcription polymerase chain reaction (RT-PCR) with ToMMV specific primers, ToMMVMPF (5'-ATGGCTCTAACTGTTAGTGGT-3') and ToMMVMPR (5'-TTAATACGAATCAGATCCCGCG-3'), which were designed based on the movement protein gene sequence of ToMMV YYMLJ isolate (KR824950). ToMMV was detected in 13 symptomatic samples (11 cherry tomato and 2 green pea plants) with a total detection rate of 3.29%. An 807-bp fragment was amplified from 2 out of 86 pea samples showing foliar chlorosis, mosaic, malformation and necrosis symptoms, and the amplicons were cloned and sequenced. Sequences obtained from the two pea samples were identical. Therefore, one sequence was deposited in the GenBank (accession no. MW561348). BLAST search result showed that the nucleotide sequence had the highest identity of 99.88% with the ToMMV TiLhaLJ isolate (KR824951). The ToMMV infection on the two pea samples was also verified by dot-enzyme immunoassay (Dot-ELISA) using ToMMV monoclonal antibody (kindly provided by Dr. Jianxiang Wu, Zhejiang University, China). To determine the pathogenicity of ToMMV on pea, ToMMV infectious cDNA clone was used to inoculate into 3-week-old healthy pea plants via an Agrobacterium-mediated method (Tu et al. 2021). Mottle or slight mosaic, chlorosis and malformation symptoms were observed on the upper leaves of the 8 out of 12 inoculated plants 8 days postinoculation, and ToMMV could be detected by RT-PCR from the 8 symptomatic plants but not from the asymptomatic and healthy control pea plants with the above described ToMMV specific primer pair. ToMMV has now only been detected in Mexico, China, Spain, USA, Israel and Australia on tomato, pepper and eggplant (Webster et al. 2014; Turina et al. 2016; Ambrós et al. 2017; Lovelock et al. 2020). To the best of our knowledge, this is the first report of natural infection of ToMMV in pea as well as the natural infection of ToMMV on plants outside of the family Solanaceae. The result also implies that ToMMV has a potential risk to more crops in the field. Since pea, pepper and tomato are economically important cash crops in China, proper virus management strategies for the cultivation of these crops should be adopted.

Background Tomato mottle mosaic virus (ToMMV) is a recently identified species in the genus Tobamovirus and was first reported from a greenhouse tomato sample collected in Mexico in 2013. In August 2013, ToMMV was detected on peppers (Capsicum spp.) in China. However, little is known about the molecular and biological characteristics of ToMMV. Methods Reverse transcription-polymerase chain reaction (RT-PCR) and rapid identification of cDNA ends (RACE) were carried out to obtain the complete genomic sequences of ToMMV. Sap transmission was used to test the host range and pathogenicity of ToMMV. Results The full-length genomes of two ToMMV isolates infecting peppers in Yunnan Province and Tibet Autonomous Region of China were determined and analyzed. The complete genomic sequences of both ToMMV isolates consisted of 6399 nucleotides and contained four open reading frames (ORFs) encoding 126, 183, 30 and 18 kDa proteins from the 5’ to 3’ end, respectively. Overall similarities of the ToMMV genome sequence to those of the other tobamoviruses available in GenBank ranged from 49.6% to 84.3%. Phylogenetic analyses of the sequences of full-genome nucleotide and the amino acids of its four proteins confirmed that ToMMV was most closely related to Tomato mosaic virus (ToMV). According to the genetic structure, host of origin and phylogenetic relationships, the available 32 tobamoviruses could be divided into at least eight subgroups based on the host plant family they infect: Solanaceae-, Brassicaceae-, Cactaceae-, Apocynaceae-, Cucurbitaceae-, Malvaceae-, Leguminosae-, and Passifloraceae-infecting subgroups. The detection of ToMMV on some solanaceous, cucurbitaceous, brassicaceous and leguminous plants in Yunnan Province and other few parts of China revealed ToMMV only occurred on peppers so far. However, the host range test results showed ToMMV could infect most of the tested solanaceous and cruciferous plants, and had a high affinity for the solanaceous plants. Conclusions The complete nucleotide sequences of two Chinese ToMMV isolates from naturally infected peppers were verified. The tobamoviruses were divided into at least eight subgroups, with ToMMV belonging to the subgroup that infected plants in the Solanaceae. In China, ToMMV only occurred on peppers in the fields till now. ToMMV could infect the plants in family Solanaceae and Cucurbitaceae by sap transmission.