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First Report of Shallot Virus X and Shallot Latent Virus on Shallot (Allium cepa var. aggregatum) in China

Allium species are economically important crops in the world. Viruses are among the most important pathogens affecting their yield (Katis et al. 2012). Only onion yellow dwarf virus (OYDV) has been found in shallot in China (Su et al. 2019). Shallot virus X (SVX, genus Allexivirus, family Alphaflexiviridae) and shallot latent virus (SLV, genus Carlavirus, family Betaflexiviridae) were reported in many Allium species (Chodorska et al. 2014; Kanyuka et al. 1992). In 2018, symptoms consisting of severe leaf twisting, yellowing, and plant stunting were observed on shallot (Allium cepa var. aggregatum) in Jilin Province, China. The symptoms were found in all shallot fields investigated (27), and symptoms were noticed in 20 to 75% of plants. A total of 18 symptomatic plants were collected from nine shallot fields and tested by double antibody sandwich ELISA kit for SVX (DSMZ RT-1042) and TAS-ELISA kit (DSMZ RT-0514-0512/1) for SLV. SVX was detected in two out of 18 shallot samples, and SLV was detected in 15 out of 18 shallot samples. All SVX-positive samples were also SLV positive. The three SVX- and SLV-negative plants were positive for OYDV (using methods as Su et al. 2019) with chlorosis and yellowing. Presence of SVX and SLV in ELISA-positive shallot plants was further confirmed by reverse transcription PCR (RT-PCR). Total RNAs of 18 plant samples were extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), reverse transcription and PCR were carried out with the PrimeScript II first Strand cDNA Synthesis Kit (Takara, Japan) and Premix Taq (Ex Taq version 2.0) (Takara). The SVX coat protein (CP) specific primer pair (svxu, 5′-ATGAACGAAGASGATTTGAATCG-3′; svxd, 5′-ARATCGTGKGGATGCATCAGAA-3′) and the SLV CP specific primer pair (slvu, 5′-GGTTCACTTTAGGTTTACAGC-3′; slvd, 5′-GYTTGATCAACATCGATTCTC-3′) designed to amplify complete CP gene of each virus were used for amplification. All SVX ELISA-positive shallot plants yielded an amplicon of the expected size (804 bp), and the SLV-positive plants yielded a 916-bp product each. One RT-PCR product per virus species were sequenced after T vector ligation with the pMD 18-T Vector Cloning Kit (Takara) and submitted to GenBank (accession nos. MK331812 for SVX and MK331810 for SLV). Pairwise comparison of the sequences with other isolates of these viruses available in GenBank showed that CP gene of SVX isolate shared the highest nucleotide/amino acid identities of 86.1/ 94.7 and 86/95.4% with the CP gene of SVX isolates NZ (EU835197) and Russian (JX310755) from New Zealand and Russia, respectively. The CP gene of SLV isolate Nongan-2 shared the highest nucleotide/amino acid identities of 83.0/95.6 and 83.0/95.6% with the CP gene of SLV isolate Xixia (AJ307034) and SLV isolate SLV-WOST87 (LC279526) from China and Japan, respectively. To our knowledge, this is the first report of SVX and SLV infecting shallot in China. All plants with SLV and SVX coinfection showed severe dwarfism symptoms compared with plants with a single infection of SLV. The spread of both viruses could threaten shallot production. Shallot asexual reproduction has been applied for more than 70 years in Jilin and Heilongjiang Provinces; virus detection and virus-free bulb application can avert further spread. Further investigation is necessary to prevent the spread of these pathogens to new locations and to new hosts (Naderi Saffar et al. 2013) in China.