First Detection of Pepper Chat Fruit Viroid (PCFVd) in Commercial Pepper Greenhouses Using Molecular Diagnostics

Abstract

Pepper is an economically important vegetable crop cultivated extensively under greenhouse conditions; however, its production is threatened by several emerging pathogens, including viroids that can cause severe yield and quality losses. Pepper Chat Fruit Viroid (PCFVd) is a highly infectious viroid associated with symptoms such as stunted growth, leaf distortion, reduced fruit size, and malformed pepper fruits. The present study was conducted to investigate and confirm the occurrence of PCFVd in commercial pepper greenhouses using molecular diagnostic techniques. Field surveys were carried out in major greenhouse production areas, where symptomatic pepper plants exhibiting chlorosis, deformation, and fruit abnormalities were sampled for laboratory analysis. Total RNA was extracted from collected plant tissues and subjected to reverse transcription polymerase chain reaction (RT-PCR) using viroid-specific primers targeting conserved regions of the PCFVd genome. Amplified products were sequenced and analyzed using BLAST and phylogenetic approaches to confirm molecular identity and determine genetic relationships with previously reported isolates. The results confirmed the presence of PCFVd in symptomatic greenhouse pepper plants, while asymptomatic samples showed negative amplification. Sequence analysis revealed high nucleotide similarity between the detected isolates and known PCFVd sequences reported from other geographic regions, confirming the identity of the viroid. Phylogenetic analysis demonstrated close evolutionary relationships among the isolates, suggesting possible dissemination through infected planting material or greenhouse production practices. This investigation represents the first detection of Pepper Chat Fruit Viroid in commercial pepper greenhouse systems within the studied region. The findings highlight the importance of early molecular diagnosis and continuous surveillance for preventing the spread of viroid diseases in protected cultivation systems.