Identification and characterization of viruses infecting tobacco (Nicotiana tabacum L.) in South Africa.

Abstract

Tobacco (Nicotiana tabacum L.) is one of the most economically important crops which contributes more than R17 billion to the national GDP of South Africa each year. Production, yield and quality of tobacco have been seriously affected by a number of factors including the emerging and recurrent plant viruses. A number of viruses are known to infect tobacco resulting in substantial yield losses. Orthotospoviruses belong to the group of emerging viruses which have shown to spread rapidly over the last decade. These viruses are among the top ten pathogens known to infect a number of crops worldwide. In 2019, large thrips populations accompanied with severe necrotic, ringspot and chlorotic symptoms typical of Orthotospovirus infections were observed in tobacco fields across South Africa (SA). Plant material showing these virus-like symptoms were collected from the three provinces of Limpopo, Northwest and Western Cape for laboratory analysis. A total of 22 leaf samples of different cultivars exhibiting orthotospovirus-like symptoms were first tested using Enzyme-Linked Immunosorbent Assay with Orthotospovirus specific antibodies, of which 19 of the 22 tested positive. The same samples were further tested with a reverse-transcriptase polymerase reaction (RT-PCR) using 2 sets of primers, firstly with orthotospovirus genericprimers, and then with tomato spotted wilt virus (TSWV) specific primers. RT-PCR results showed that 19 of the 22 samples tested positive for Orthotospoviruses and 11 of the 22 samples tested positive for TSWV. Nineteen positive sample PCR products PCR products of the positive samples were sent for Sanger sequencing. The sequences obtained were subjected to Basic Local Alignment Search Tool (BLAST) and two orthotospoviruses were detected; TSWV and groundnut ringspot virus (GRSV). Eleven of the 19 sequences matched with TSWV and eight matched with GRSV. According to our knowledge, this is the first report of GRSV infecting tobacco in SA. To determine if the symptoms observed on the tobacco plants were due to mixed virus infections, samples were subjected to Next Generation Sequencing (NGS). For this technique the total RNA was extracted from the frozen symptomatic leaf samples using a Quick-RNA™ Plant Miniprep kit (ZYMO Research, USA). To save costs, all the extracted RNA from the 22 samples was mixed together into one sample and sent to Agricultural Research Council (Biotechnology Platform) for NGS library preparation and sequencing. The NGS data was analysed using the online pipeline software, Genome Detective Virus Tool Version 1.133. The results showed that 11 other plant viruses from different genera, namely; tobacco mosaic virus (TMV), west African asystasia virus 1 (WAAV1), potato virus Y(PVY), tobacco vein clearing virus (TVCV), tomato leaf curl Uganda virus (ToLCUV), petunia vein clearing virus (PVCV), cucumber mosaic virus (CMV), beet mosaic virus (BMV), beet western yellows virus (BWYV), beet cryptic virus 2 (BCV2), and beet cryptic virus 3(BCV3). These viruses that co-infected tobacco with orthotospoviruses resulting in the severe symptoms observed in the field. NGS analysis also detected the presence of a third orthotospovirus; tomato chlorotic spot (TCSV) in the samples. NGS also was able show the presence of partial and complete sequences for the viruses mentioned above with a coverage between 70-100%. The phylogenetic analysis was done to determine the relationship of each these viruses with other nucleotide sequences of the same species or genus from GENEBANK. Viruses were grouped into clusters according to the sequences of the closest relatives in the genus, and they had a nucleotide identity ranging from 56 to 99 percent to their closest species, which indicates the occurrence of some new isolates The information presented in this study shows that viruses constitute a significant threat to the economic production of tobacco in SA. Several emerging viruses from different genera that infect tobacco in South Africa were detected using NGS. Most of the viruses detected by NGS are being reported on tobacco in SA for the first time. This study demonstrates the importance and effectiveness of using NGS for plant virus identification without any prior knowledge or based on the symptoms of the virus. Unlike other methods used to identify viruses infecting plants, NGS can detect mixed virus infections. The accurate identification of all viruses infecting tobacco at any given time is crucial for developing effective and sustainable control strategies.