Strawberry, a New Natural Host of Brassica Yellows Virus in China

Abstract

Brassica yellows virus (BrYV; genus Polerovirus, family Solemoviridae) has an icosahedral spherical virion with a positive-sense single-stranded RNA genome and it is distinguished from turnip yellows virus (TuYV) based on differences in ORF0 and ORF5 (Xiang et al., 2011). To investigate the occurrence and distribution of viruses infecting strawberry (Fragaria ananassa) in the main production areas in China, a survey of nine greenhouses (667 m2 each) was conducted in the cities of Yantai and Beijing, China in August 2020. About 1% of strawberry plants in each greenhouse showed virus-like symptoms of chlorotic spots; 89 symptomatic leaf samples were randomly collected for virus testing. Total RNA was extracted from a pool of eight samples of four different cultivars (Hokowase: 2, Mibao: 2, Sagahonoka: 2, Monterey: 2) from Yantai using RNAprep Pure Plant Plus Kit (TianGen, China). A cDNA library was constructed by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) after ribosomal RNA-depletion using an Epicentre Ribo-Zero™ rRNA Removal Kit (Epicentre, USA). High-throughput sequencing was done on Illumina Hiseq 4000, generating 70,931,850 high-quality 150 bp paired-end reads. Clean reads were de novo assembled by Trinity (v2.2.0) and the resulting contigs were screened by BLASTn and BLASTx against GenBank database as described previously (Grabherr et al., 2013). A total of 1,432,164 high-quality reads unmapped to the strawberry genome were obtained and assembled into 93 contigs (ranging from 33 to 8,031 nt). Seven of these contigs (277 to 1,254 nt) shared 98.2 to 100% nt identities with BrYV-A (accession no. HQ388348) and covered 89.5% of the genome of BrYV-A. Subsequent analyses indicated the presence of Strawberry pallidosis-associated virus and Strawberry mottle virus in the analyzed sample, both have been reported in strawberry in China (Shi et al., 2018; Fan et al., 2021). To confirm BrYV infection, total RNA was isolated from the eight samples used for HTS and reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers (CP and rtp, Supplementary Table 1) designed based on the assembled contigs. PCR products with expected sizes (587 and 609 bp) were observed in one sample (cv. Mibao). BLASTn analysis indicated that the amplicons (accession no. MW548437 and MW548438) shared 98.6% and 99.3% nt identity with BrYV-A, respectively. To obtain the complete sequence of the putative BrYV isolate, the gaps were bridged and the terminal sequences were determined using 5ʹ and 3ʹ RACE kits (Clontech, China) based on the assembled contigs. The complete genome sequence of the putative BrYV isolate has a length of 5,666 nt (accession no. MZ666129) and shares more than 94.3% nt identities with other BrYV isolates. Phylogenetic analysis indicated that the isolate grouped closely with BrYV and further from TuYV (Figure S1). In addition, 11 samples (cv. Benihoppe) of the remaining 81 symptomatic strawberry samples tested positive for BrYV by RT-PCR with the two pairs of primers mentioned above. The sequences (accession no. MZ407232 and MZ407233) revealed 99.5% and 99.3% nt identities with MW548437 and MW548438. To the best of our knowledge, this is the first report of natural infection of BrYV in strawberry plants. Our findings expand the host range of BrYV, but disease association is difficult to establish due to presence of mixed infection and non-fulfillment of Koch's postulates.

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR

“…Previously, we discovered that single or mixed virus infection is common in strawberries [ 20 ]. In a previous study, BrYV was discovered for the first time in strawberry plants from Shandong province, China [ 8 ]. In the present study, the complete genome sequences of BrYV-MB and BrYV-HY were obtained, and the molecular features were characterized.…”

“…Then, 8 strawberry leaves (Hokowase: 2, Mibao: 2, Sagahonoka: 2, Monterey: 2) with typical viral symptoms were pooled to construct a cDNA library for HTS. RNA-Seq and bioinformatics analysis were performed as described previously [ 8 ]. Based on the assembled contigs and the complete nucleotide sequences of BrYV in GenBank, three primer pairs (BrYV-gap1-F/BrYV-gap1-R, BrYV-gap2-F/BrYV-gap2-R, and BrYV-gap3-F/BrYV-gap3-R) were designed for amplifying three bridged segments corresponding to the complete genomes of BrYV-HY and BrYV-MB, respectively.…”

“…Currently, approximately 30 viruses and 1 viroid have been reported to infect strawberry [ 7 ]. Recently, we found that strawberry is a natural host of brassica yellows virus (BrYV) in China for the first time [ 8 ]. BrYV is widespread in China, Japan, and South Korea, and it primarily infects Cruciferous crops such as cabbage, rappini, and radish, causing leaf yellowing, curling, and necrosis in the field [ 9 , 10 , 11 ].…”

“…BrYV is widespread in China, Japan, and South Korea, and it primarily infects Cruciferous crops such as cabbage, rappini, and radish, causing leaf yellowing, curling, and necrosis in the field [ 9 , 10 , 11 ]. BrYV is a phloem-restricted virus; it may enter the host phloem by aphid transmission and Agrobacterium infiltration, but it cannot be transferred mechanically [ 8 , 12 ].…”

“…Previously, we found that BrYV can naturally infect strawberry in China [ 8 ], but its molecular characteristics remain largely unknown. Viral infectious cloning is the basis for RNA virus research constructed using reverse genetic technology [ 14 ].…”