First Report of Leek Yellow Stripe Virus Infecting Allium cepa in China.

Onion (Allium cepa), a member of the genus Allium, is widely cultivated throughout the world including China (Zhang et al. 2022). In July 2022, stunted onion (A. cepa 'Weiwang') plants showing typical symptoms of yellow stripe and leaves distortion (Fig. S1) were observed in a vegetable garden in Hohhot, Inner Mongolia, China. The garden is approximately 0.24 ha with around 20,000 onion plants, out of which 140 plants were symptomatic. Diagnosis of the symptomatic plants using negative stain electron microscopy revealed the association of long flexuous virus particles measuring about 11 to 12 nm × 820 to 1000 nm (Fig. S2), which was suggestive of the presence of potyvirus (Chen et al. 2002). Subsequently, the pathogen was identified as the leek yellow stripe virus through RT-PCR combined with Sanger sequencing as described below. The total RNA of each sample was extracted using the MiniBEST plant RNA extraction kit (TaKaRa, Dalian, China), serving as template for synthesis of cDNA using the ABScriptIII RT master mix (ABclonal Biotechnology, Wuhan, China). We then amplified a fragment at the 3' terminus of LYSV using a M5 Hiper superluminal mix (Mei5 Biotechnology, Beijing, China) with the primer pair LYSV-F / LYSV-R (Santosa and Ertunc 2020) which flank the partial NIb gene, the complete coat protein gene and partial 3' untranslated region of LYSV. A unique PCR product of about 1 kb was seen for 10 out of the 140 samples. Five out of the 10 PCR products were randomly selected and cloned using a Zero Background pTOPO-TA cloning kit (Aidlab Technologies, Beijing, China) and E. coli JM109 competent cells were then transformed. Positive colonies were screened by PCR detection of the insert fragments using the primers LYSV-F/-R, and the inserts were sequenced at BGI Genomics (Beijing) using the M13(-21) Forward and M13 Reverse primers. All the obtained sequences were 1032 nt in length, and shared nucleotide sequence identities of 99.2% to 100% (two out of the five sequences were identical to each other). The query sequences were submitted to BLASTn to retrieve homologous sequences from NCBI GenBank databases, and the results showed that the four sequences were homologous to LYSV, suggesting the occurrence of LYSV on onions in Inner Mongolia, China. The sequences were then deposited in GenBank under accession numbers of OQ969953-56, named LYSV isolate Hohhot-1, -2, -3, and -4. In comparison with other published LYSV isolates, the LYSV Hohhot-1, -2, -3, and -4 had the highest nucleotide sequence identity of 87.23%, 86.97%, 87.33%, and 87.23% with LYSV G66 (GenBank accession no. MN059493), respectively, which infects garlic in China. Phylogeny analysis was performed based on 41 complete sequences of the CP gene of LYSV, including the four in this work and another 37 from GenBank of which six isolates were discovered in onions in Turkey (MN070124, MN070126, MN070130, MN070131, MN864794 and MN864795) and the others 31 isolates were from garlics or leeks in 15 different countries (Argentina, Australia, Brazil, China, Ethiopia, Germany, India, Iran, Japan, Mexico, Netherlands, New Zealand, Serbia, South Korea, and Spain), while the CP gene of onion yellow dwarf virus (AJ510223) was employed as an outgroup reference. The tree was reconstructed using the neighbor-joining method of MEGA11 with a bootstrap value of 1,000 replicates. On the tree (Fig. S3), the LYSV Hohhot-1, -2, -3, and -4 were closely related to each other and were distinct from other LYSV isolates including the six isolates in onions in Turkey, suggesting a specific genetic variation of the LYSV isolates in Hohhot. According to Santosa et al. (2023), LYSV Hohhot-1, -2, -3, and -4 were within the S-type lineage. This was the first record of LYSV infecting onions in China, expanding the natural host range of LYSV in China, which offered important information for the management of onion diseases.