Seed transmission and genome characterization of tobacco streak virus in marigold: Development of qPCR and RT-LAMP assays for host and vector detection

Abstract

Marigold plants (30 samples) exhibiting virus-like symptoms were collected from diverse agro-climatic regions of Karnataka, Maharashtra, and Tamil Nadu to identify the causal agent. Initial serological assays indicated a positive reaction to tobacco streak virus (TSV). Infectivity was confirmed by mechanically inoculating symptomatic leaf extracts onto cowpea indicator plants (cv. C-152), which developed characteristic symptoms, demonstrating biological activity of the virus. For molecular confirmation, total RNA was extracted from eight representative plant samples (seven marigold and one cowpea), two seed samples, and five thrips collected from infected fields. RT-PCR with coat protein (CP) gene-specific primers consistently amplified a ∼700 bp fragment from marigold, cowpea, and thrips samples. Sequencing of cloned amplicons revealed >97 % nucleotide identity with Indian TSV isolates infecting okra, cotton, groundnut, sunflower, and cucumber. Given the frequency of TSV incidence and the extent of marigold cultivation, one representative isolate was selected for complete genome sequencing. Using segment-specific primers, RNA1, RNA2, and RNA3 were amplified, cloned, and sequenced. Comparative genome analysis showed >97 % identity with Indian isolates and with TSV isolates from squash and pumpkin in the United States, suggesting cross-host adaptation. Sequencing of the mitochondrial cytochrome oxidase I (mtCOI) gene identified Thrips tabaci as the vector transmitting TSV. Furthermore, quantitative PCR (qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed and validated for rapid and sensitive TSV detection in marigold tissues and thrips. This study reports the first complete genome sequence of marigold-associated TSV isolate, confirming its dual transmission via seeds and thrips, and providing critical insights into its molecular epidemiology and threat to ornamental crops in India.