Primary distribution and RT-PCR-based detection of citrus dwarfing viroid (CDVd) in citrus cultivars of Pakistan

Viroid diseases have emerged as a significant threat to citrus orchards globally. In Pakistan, viroid infections have rapidly spread across different citrus cultivar, posing serious challenges to the survival of orchards. This study encompasses the determination of the prevalence and detection of Citrus dwarfing viroid (CDVd) in both reported and newly introduced citrus cultivars in Pakistan using RT-PCR. RNA was extracted from 122 infected citrus samples and examined for the presence of viroids following the successful optimization of newly designed CDVd primers (CDVd AF1/AR1). This led to the confirmation of CDVd in 72 samples (59.01%).. Biological indexing was performed using T-grafting methods on one-year-old healthy Arizona 861-S1 Etrog citron plants under controlled conditions to confirm the presence of CDVd infection in collected infected budwoods of different citrus cultivars. This led to the detection of CDVd along with CVd-V and CBLVd, confirming mixed infections. Sequencing analysis of PCR-amplified products confirmed that the plants were infectedwith CDVd. Notablythis study identified ‘Palestine sweet lime’ as a noval host of CDVd in Pakistan, marking the first recorded instance. These findings underscoure the need to develop strategies for diagnosing and preventing the further spread of CDVd in citrus orchards to ensure sustainable citrus production.