First Report of Tomato Bushy Stunt Virus Naturally Infecting Sugar Beet in the United States

Abstract

Sugar beet (Beta vulgaris L.) is an important crop grown for its sucrose content used in sugar production around the world. Tomato bushy stunt virus (TBSV) is an RNA virus that belongs to the Tombusvirus genus of the family Tombusviridae (Hearne et al., 1990). The virus was first isolated from tomato, and it is known to infect a wide range of plants (Smith, 1935; Martelli et al., 1988; Hafez et al., 2010). In 1980, a natural infection of TBSV was reported in sugar beet leaves with chlorotic and necrotic ring spots and line pattern symptoms based on serological affinity to TBSV anti-sera in Czechoslovakia (Novak and Lanzova, 1980). In March 2021, sugarbeet plants showing stunted and bushy growth with yellowing and necrotic leaves were observed in a production field in the Imperial Valley of California. Harvested roots exhibited stunted and abnormal growth compared to roots from healthy plants (sFig. 1A). These symptoms prompted a screen for potential infection by TBSV. Root-tissue harvested from the symptomatic sugar beet was initially screened using a TBSV double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA; Agdia, Inc., Elkhart, IN), which reacted positive for TBSV. To obtain the full-length sequence of TBSV and potentially other viruses in the sample, total RNA isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) from the root-tissue was subjected to high-throughput sequencing (HTS). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA) and sequenced using Illumina NovoSeq 6000 paired-end platform (Novogene, Sacramento, CA). A total of 52 million reads were obtained after removing the adapters and reads mapping to the host genome. These high-quality reads were de novo assembled into 75,891 contigs that are larger than 500 base pairs using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The resulting contigs were searched for matching sequences to known viruses using the NCBI non-redundant database. A single contig of 4770 nts representing the full-length genome of TBSV was generated (Accession number OP477335), which showed 100% coverage to previously reported TBSV isolates ‘statice’ (AJ249740.1) and ‘nipplefruit’ (AY579432.1) with 92.19% and 91.25% nucleotide sequence identities, respectively, and thus confirming the presence of TBSV in sugar beet root-tissue. However, it showed 74% coverage with only 87% nucleotide identity to a previously reported Lettuce necrotic stunt virus (LNSV) from sugar beet, a tombusvirus that was re-classified as Moroccan pepper virus (MPV) due to high degree (>97%) of sequence identity (Obermeier et al., 2001; Wintermantel and Anchieta, 2012; Wintermantel and Hladky, 2013). The coat protein is conserved within species in tombusvirus, and it plays a significant role by providing serological relationships to tombusvirus taxonomy. The coat protein of TBSV-isolate of this study shared 98.45% and 96.91% identities at amino acid level with TBSV ‘nipplefruit’ (AY579432.1) and TBSV ‘statice’ (AJ249740.1) isolates, respectively. In contrast, it showed only 61.56% identity with the coat protein of MPV as shown in the phylogenetic tree indicating that the TBSV-isolate reported here is different from MPV (sFig. 2). To confirm the presence of TBSV, reverse-transcription (RT)-PCR was performed using the total RNA isolated from the root-tissue with primers (VR306: 5’-CGCTCACGAGCCCAGCATCCTTGA-3’ and VR297: 5’-ACACCGCCACAGGAGCCATGATTG-3’) designed based on the HTS data to amplify a portion of the TBSV genome. Sequencing of the RT-PCR product confirmed the presence of TBSV sequence with 99.1% identity to the TBSV-isolate identified in this study. Further, mechanical inoculation of total RNA isolated from the symptomatic sugar beet roots produced local lesions and systemic necrosis symptoms on the leaves of Chenopodium quinoa (sFig. 1B). Sequencing of the amplicon obtained using RT-PCR with primers VR306 and VR297 confirmed the presence of TBSV in C. quinoa. In addition to TBSV, several viral contigs representing Beet necrotic yellow vein virus were identified in the root-tissue indicating mixed infection in the field. To our knowledge, this is the first report that documents the occurrence of TBSV in sugar beet in the United States. Since TBSV is a soil-borne virus, our findings indicate the need for further studies focused on the frequency and coexistence of the TBSV with BNYVV in sugar beet production fields to understand the disease complexity resulting from potential mixed infections.