First Report of Potato Spindle Tuber Viroid in Vegetative <i>Capsicum annum</i> and Its Effect on Host Metabolites

ABSTRACT In recent years, Potato spindle tuber viroid (PSTVd), a member of the genus Pospiviroid, has been increasingly detected in a wide range of vegetatively propagated and ornamental plant species. This pilot study was conducted to detect the presence of PSTVd in Capsicum annuum L. (chilli) plants and to analyse the volatile organic compounds (VOCs) emitted by infected plants using gas chromatography–mass spectrometry (GC–MS). Field surveys were carried out in various nurseries located in Pattoki, District Kasur, Pakistan, to identify symptomatic chilli plants exhibiting leaf curling, yellowing and spindle‐shaped deformities. Suspected samples were collected from multiple nursery sites, encompassing a range of developmental stages from recently grafted seedlings to fruit‐bearing plants. For molecular diagnostics, a modified Trizol‐based RNA extraction protocol was employed to enhance efficiency and conserve reagents. Total RNA from symptomatic leaves was reverse transcribed to cDNA and subjected to sequencing. Sequence analysis revealed that 75 out of 100 samples exhibited up to 99% sequence similarity with known PSTVd isolates in GenBank, with the predominant local isolate submitted under accession number MK977647. Comparative analysis suggests that the introduction and spread of PSTVd in chilli crops may be associated with proximity to infected ornamental species (e.g., MN055490) and Solanaceous crops such as potato and tomato. As far as we are aware, this study provides the first documented evidence of PSTVd infection in chilli plants in Pakistan. These findings underscore the necessity for continued surveillance and the development of integrated management strategies to mitigate the spread of this economically significant viroid in vegetatively propagated crops.