Sunflower necrosis disease from India is caused by an ilarvirus related to <i>Tobacco streak virus</i>

Abstract

A serious disease of sunflower (Helianthus annuus) with putative virus aetiology was found occurring in all major sunflower-growing regions of India. Symptoms of this sunflower necrosis disease (SND) comprised chlorotic and necrotic ringspots and leaf distortion. A general leaf and stem necrosis extending to midveins, petioles and flower bracts eventually results in stunting and dieback, especially when plants become infected in the early stages of development. Ilarvirus-like particles were detected by electron microscopy in crude sap of SND-affected sunflower, and in Chenopodium quinoa plants inoculated with leaf extracts prepared from SND-affected sunflower plants. In addition to several other herbaceous virus indicator plants, groundnut, cowpea and cotton, significant crops in India, also became infected. Back-transmission to healthy sunflower seedlings with leaf extracts of systemically infected indicator plants resulted in identical symptoms of SND, confirming the ilarvirus-like component as the causative agent of sunflower necrosis disease. An antiserum (DSMZ AS-0615, Braunschweig, Germany) raised against purified virus preparations reacted well in DAS–ELISA and proved reliable for field diagnosis of Sunflower necrosis virus (SNV). In a comparative serological study using plants infected with other ilarviruses, a reaction was demonstrated only with Tobacco streak virus (TSV). This serological relationship was confirmed by Western blot analysis and by immunosorbent electron microscopy decoration assays using SNV and TSV antisera in reciprocal tests. In RT–PCR, using oligonucleotide primers deduced from conserved sequences within TSV RNA3 and flanking the entire coat protein region, an approximately 1000 bp dsDNA fragment was amplified from SNV-infected sunflowers. Sequence analysis of cloned SNV PCR fragments revealed nucleotide identities of approximately 90% with TSV RNA3 (EMBL accession number X00435) and a coat protein amino acid homology between SNV and TSV of more than 90%. The variability of nucleotide sequences retrieved from three sunflower isolates from different locations was less than 3%. These experimental data unequivocally prove that the virus causing sunflower necrosis disease in many sunflower varieties and in many different sunflower-growing regions of India is a strain of Tobacco streak virus. It is now important to establish whether TSV is associated with diseases of other significant crops, such as groundnut, in India.