First Report of Lettuce Chlorosis Virus Infecting Chinese Cabbage in China.

Abstract

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a cruciferous vegetable of significant agricultural importance in China, valued for its high yield and nutritional benefits (Yun et al., 2021). However, viral diseases pose a serious threat to its production, affecting both yield and quality (Wang et al., 2024). In November 2021, leaf samples exhibiting viral-like symptoms, including chlorosis, leaf curling, and stunting, were collected from a Chinese cabbage field in Baoding, Hebei Province, China. Total RNA extracted from symptomatic leaves of 8 Chinese cabbages with viral symptoms using TRIzol Reagent (Invitrogen) was pooled to generate composite samples. These combined RNA samples were then processed for library construction and analyzed by high-throughput sequencing (HTS) on the Illumina MiSeq platform to identify viral pathogens. A total of 15,872,109 raw reads were generated and has been deposited in Sequence Read Archive (SRR31969092), of which 15,681,883 (98.80%) were retained as clean reads after quality filtering. Subsequently, 7,504,572 reads were mapped to the reference genome (http://www.bioinformaticslab.cn/EMSmutation/home/). Small RNAs (sRNAs) ranging from 18 to 26 nucleotides in length were isolated and assembled into 1,079 contigs using Velvet and PFOR. Contigs were annotated and classified using the Virus RefSeq Nucleotide and Virus RefSeq Protein databases, with sequence alignment performed using BLAST. Analysis revealed the presence of lettuce chlorosis virus (LCV), a member of the genus Crinivirus, family Closteroviridae (Salem et al.,2009). The bipartite single-stranded RNA genome comprises RNA1 (8.59 kb, NC_012909.1) encoding 8 putative proteins including RNA-dependent RNA polymerase (RdRp), and RNA2 (8.56 kb, NC_012910.1) containing 7 open reading frames (ORFs) such as the coat protein (CP) gene. A total of 996 sRNAs were mapped to various positions of the LCV genome, covering 7.11% of the viral genomic sequence. Through BLAST, we found that the similarity range of the viral sequences to LCV isolate CN is from 93.33% to 100%. Specifically, LCV RNA1 and RNA2 were identified with 272 and 219 mapped reads, respectively. To confirm the presence of LCV, RT-PCR was performed using specific primers (F-cp: 5'-ATGGGTGATAGCAAAGAAAC-3', R-cp: 5'-TTATTTACTGCAACCCCCTG-3') targeting the CP gene. A 753-bp fragment of the CP gene was amplified and sequenced. BLASTn analysis showed 98.7% nucleotide sequence identity with an LCV isolate CN segment RNA2 on Periwinkle (accession no. KY430286.1). To the best of our knowledge, this is the first report of LCV infecting Chinese cabbage worldwide, and also the first report of LCV in the region north of the Yangtze River in China (Zhao et al., 2018) (Tian et al., 2018). So far, the hosts of the LCV discovered in China include Periwinkle and Madagascar periwinkle [Catharanthus roseus (L.)] and Tobacco plants (Nicotiana tabacum cv. Samsun NN). These findings expand the known host range of LCV and highlight its potential threat to cruciferous crops, necessitating enhanced surveillance and resistance breeding strategies.