Specific detection of Malus- and Pyrus-infecting viroids by real-time reverse-transcription quantitative PCR assays.

Viroids present a number of issues for their detection and diagnosis because of the absence of symptom expression in many hosts and their low titers in infected plants. However, quarantine programs rely on symptom observations and routine diagnostic testing to reduce the risk of spreading viroid-infected materials to situations where they might affect crop health and production. Sensitive, accurate, and specific assays for viroid detection from both asymptomatic and symptomatic hosts are necessary for managing viroids in post-entry quarantine and certification schemes. The aim of this study was to develop and optimize superior assays based on the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for the specific detection of apple hammerhead viroid (AHVd), apple scar skin viroid (ASSVd) and pear blister canker viroid (PBCVd). The real-time RT-qPCR assays thus developed detected a greater range of viroid isolates and with greater sensitivity than the current endpoint RT-PCR assays, down to 101 copies per reaction without any amplification of the non-target viroid or virus sequences tested.

Development of RT-qPCR assays for the detection and quantification of three carlaviruses infecting hop.

American hop latent virus (AHLV), hop latent virus (HLV) and hop mosaic virus (HMV) infect members of the Humulus genus worldwide, but very little is known of the biology and etiology of these viruses. A better understanding of these viruses from the molecular level to their economic impact relies on efficient diagnostic assays. Therefore, in this study we developed reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays for the detection of AHLV, HLV, and HMV through an alignment of representative sequences from the National Center for Biotechnology Information (NCBI) database. These assays demonstrated unambiguously their high sensitivity by detecting the respective targets from as low as 102 copies of transcripts per reaction without any amplification from non-targets.

Development of real-time RT-PCR assays for two viruses infecting pome fruit.

Apple stem grooving virus (ASGV) and Apple green crinkle-associated virus (AGCaV) negatively impact production, maintenance, and distribution of apples and other Malus species world-wide. Due to the increasing diversity of isolates found by high-throughput sequencing, we have developed real-time RT-qPCR assays for these two viruses. Primers and probes were designed against alignments of representative extant sequences from around the world, and reaction conditions optimized for sensitivity and specificity. Assays were validated against a panel of virus isolates, and compared to extant endpoint RT-PCR and ELISA assays. The new real-time RT-qPCR assays showed greater detection sensitivity than extant assays and were able to detect their target viruses from different host tissues.

Diversity of three bunya-like viruses infecting apple.

High-throughput sequencing of two trees with apple decline revealed the presence of three bunya-like viruses: apple rubbery wood-associated viruses 1 and 2 (ARWaV-1, ARWaV-2) and citrus concave gum-associated virus (CCGaV), which previously had only been observed in citrus trees. The apple and citrus CCGaV isolates shared over 97% sequence identity. A global collection of apple trees was screened by RT-PCR for these viruses. Twenty-seven of 30 trees were infected with one or more bunya-like virus. Sequence data revealed some diversity among isolates but no geographic grouping. Additional work will be needed to determine if any of these viruses contribute to apple decline.