Citrus is a genus of flowering trees and shrubs in the family Rutaceae, which includes approximately 1600 species of plants (Swingle, 1943), many of which are economically important fruits such as orange, mandarin, lemon, limes, grapefruit and pomelos. The global trade of citrus each year is significant. According to the Food and Agriculture Organization (FAO), the global trade of citrus fruits and products was valued at over US$15 billion in 2019. Aotearoa New Zealand's (NZ) citrus export trade is relatively small compared to other countries, such as Spain (US$4.12 billion), South Africa (US$1.93 billion), China (US$1.21 billion) and Turkey (US$982 million) (OEC). In 2020, the value of NZ’s citrus exports was NZ$ 11.6 million (approx. US$7 million).

Citrus production is frequently impaired by diseases caused by various pathogens, including fungi, bacteria, nematodes, viruses, and viroids. More than 30 viruses and virus-like diseases have been identified as major contributors to citrus diseases such as leaf discoloration or deformation, plant stunting or decline, and fruit malformation. These diseases are mainly spread through infected plant materials and therefore, quarantine measures and strict biosecurity protocols are necessary to prevent their introduction and spread.

Hence, it is crucial to have sensitive and specific techniques for accurately detecting significant viruses that infect citrus in order to maintain high plant import standards. The detection of viruses in citrus has traditionally relied on serological methods (Djelouah et al., 2002) such as enzyme-link immunosorbent assay (ELISA). Nonetheless, the effective detection of viruses through ELISA is often hindered due to several factors, including the unavailability of virus-specific antibodies, variations in affinity towards different virus strains or variants, and the risk of false negatives due to low virus titre not being detected. To address these limitations and fulfil the need for improved specificity and sensitivity in virus diagnostics, molecular diagnostic techniques such as reverse transcription polymerase chain reaction (RT-PCR) (Barthe et al., 1998) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) (Osman et al., 2015) have been consistently adopted for the detection and differentiation of citrus viruses and their variants.

Currently, most citrus viruses and viroids are detected using RT-PCR (Ito et al., 2002, Roy et al., 2005, Choudhary et al., 2017; Ramos-González et al., 2017). The drawbacks of RT-PCR assays compared to RT-qPCR include lower sensitivity and longer time needed to obtain results. RT-PCR is a traditional endpoint detection method where the amplification is measured after the reaction is completed. This can lead to lower sensitivity as the detection is based on the endpoint analysis, which may require a higher viral load to yield a positive result. Another advantage of RT-qPCR over RT-PCR is being faster, which can significantly reduce workload. RT-qPCR can provide results in just 1 and a half hours, whereas RT-PCR requires at least 4 hours (from RT-PCR to electrophoresis analysis).

In NZ, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). For most of them, there is no published RT-qPCR method to date, including CILRV, CiLV-C2, CiLV-N, CYMV, CBLVd, CVd-V and CVd-VI. For CiLV-C and CPsV, existing RT-qPCR assays were published in 2015 (Choudhary et al., 2015, Osman et al., 2015). However, more than 461 new sequences of CiLV-C and 263 new sequences of CPsV have been published in NCBI since 2015, making it necessary to redesign the assays to ensure detection of all known isolates.