First report of tobacco streak virus infecting sunflower in Argentina

Abstract

Sunflower (Helianthus annuus) is one of the main oleaginous crops in the world and Argentina is positioned among the top sunflower seed producers worldwide (FAO, 2019). During the 2018-2019 crop season, sunflower plants showing symptoms similar to those reported for the tobacco streak virus (TSV) were observed in one field of Rivera, Buenos Aires province. The symptoms included leaf mosaic, severe necrosis of stem, petioles, leaves and pith, often progressing to complete collapse and death of affected plants (e-Xtra). Samples from symptomatic leaf tissue were collected from five plants and leaves of sunflower seedlings maintained under greenhouse conditions were used as healthy control. Leaf extracts obtained from symptomatic leaves were positive by DAS-ELISA test, using a polyclonal antibody against TSV (Bioreba, Switzerland). The average optical density of infected samples was 0.384, eight times higher than the mean plus three standard deviations of four healthy controls. Total RNA was extracted from collected samples using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and tested by reverse transcription-polymerase chain reaction (RT-PCR) with specific TSV primers (Almeida et al., 2005). Only infected samples produced expected amplicons (~1 kb). The RT-PCR products, obtained in duplicate from two symptomatic plants were purified and sequenced in both directions. The sequences were generated by individually sequencing and assembling each product to avoid assemble possible variants. The obtained sequences were assembled using the DNASTAR Lasergene SeqMan software (DNASTAR, Madison, WI) and generated a consensus 969 bp sequence, which included the gene of the 714 nt CP and a 255 nt 3? untranslated region. The obtained sequences were identical, then a single nucleotide sequence was deposited in GenBank database under the accession number MK732934, and used in the analysis. A BLASTn analysis of the obtained sequence with those available in the GenBank database showed the highest nucleotide sequence identity (91%, 78-100% query coverage) with TSV isolates from Australia (JX463336, EU871659, EU375481). Phylogenetic trees obtained by using Bayesian inference in Mr. Bayes 3.2.6 (Ronquist et al. 2012) indicated that the TSV isolate from Argentina clustered together with those from Australia (JX463336, EU871659, EU375481) and Brazil (AY354406) (e-Xtra). TSV has been associated with disease epidemics in sunflower and a range of legume and vegetable crops worldwide (Gracia and Feldman, 1974; Sharman et al. 2009; Bag et al. 2019). To our knowledge, this is the first report of the occurrence of TSV infecting sunflower crops in Argentina. According to Sharman et al. (2016), the use of tolerant germplasm is critical to minimise the risk of TSV epidemics in sunflower. This finding should be addressed in future studies of resistant-cultivars selection in Argentina.