Abstract
Citrus are affected by many viruses and viroids, some globally widespread and some restricted to particular countries or areas. In this study, we simulated the use of high throughput sequencing (HTS) and the bioinformatic analysis of small interfering RNAs (siRNA) as a pre-screening method to guide bioindexing and molecular detection to enhance the surveillance survey of some key or emerging citrus viruses, such as non-European citrus tristeza virus isolates (non-EU CTV), citrus tatter leaf virus, citrus leprosis virus, citrus yellow mosaic virus, and citrus bark cracking viroid, present in the EPPO lists, and the citrus yellow vein clearing virus. The HTS’s ability to detect other citrus viroids was also evaluated. The results demonstrate that HTS provides a comprehensive phytosanitary status of citrus samples either in single and multiple infections of viruses and viroids. It also provides effective information on citrus tristeza virus mixed infections despite not being able to identify the non-EU variants of the virus. Bioindexing checks each single virus infection but does not differentiate viroids on the Etrog citron indicator and is time-consuming. Molecular assays are valuable as confirmation tests of viruses and viroids but many pairs of primers are needed for a full screening and new or non-target pathogens remain undetected. In addition, the genomes of two isolates of the citrus yellow vein clearing virus and the citrus tatter leaf virus, detected in a sample from China, are described.
Highlights
The number of viruses and viroids affecting citrus has grown considerably in the last few decades [1]
We evaluated the use of high throughput sequencing (HTS) as a pre-screening method combined with bioindexing and molecular detection in a simulated surveillance survey of citrus virus and viroids regulated by EU phytosanitary measures (Figure 1)
The unmapped reads were analyzed to search for the presence of non-EU citrus tristeza virus (CTV) isolates, as well as CLVCV, citrus tatter leaf virus (CTLV), citrus leprosis virus (CiLV), citrus yellow mosaic virus (CYMV) and citrus bark cracking viroid (CBCVd), and other readsviruses was 19.6
Summary
The number of viruses and viroids affecting citrus has grown considerably in the last few decades [1]. Biosecurity reasons may create phytosanitary targets that are not categorized as quarantine pests and which must be included in regular surveillance programs, the aim being the early detection of detrimental viruses [3]. This requires detection and identification methods that are in line with current international standards together with a statistically sound and risk-based pest survey approach [4]. According to the categorization of citrus virus and viroid diseases in the EuroMediterranean region, the European and Mediterranean Plant Protection Organization (EPPO) has drawn up two lists of pests whose regulation is relevant for the entire EPPO region or large parts of it [5]. The satsuma dwarf virus affects mainly citrus varieties less relevant in the EPPO region and the causal agent of the citrus blight remains unknown, and no direct test is available [6]
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