Detection and persistence of citrus bark cracking viroid and other viroids in citrus peel oils for agricultural applications

Abstract

Plant-based agricultural products, like citrus peel oils, are increasingly used as sustainable alternatives to synthetic pesticides. However, in crops such as hop ( Humulus lupulus L.), where viroid infections can seriously reduce yields, there is concern that products made from infected citrus might transmit viroids, especially citrus bark cracking viroid (CBCVd). This study evaluates the risk of viroid transmission by examining CBCVd, hop stunt viroid (HSVd), and citrus exocortis viroid (CEVd) through orange oil using RNA extraction and RT-qPCR analysis. Two extraction methods were tested, with the chaotropic protocol outperforming the detergent-based approach for isolating RNA from oil matrices. Spiking experiments confirmed consistent detection of CBCVd and the plant RNA marker NAD in mixtures containing 90% RNA and 10% oil, even after seven days, indicating RNA stability in oil-rich environments. In contrast, pure oil samples showed no viroid RNA or NAD detection, suggesting limited RNA persistence in pure oil. Of 32 citrus peel samples tested, CBCVd was detected in one and HSVd in seven, but no viroid RNA or NAD was detected in the corresponding oils. These findings indicate a minimal risk of viroid transmission through orange oil; however, formulations containing surfactants or water may allow RNA to partition into aqueous phases, potentially increasing the risk. This study highlights the need for routine testing of raw materials and final citrus-based products to ensure phytosanitary safety.