Simultaneous detection of citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) by multiplex RT-PCR
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Simultaneous detection of citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) by multiplex RT-PCR Maria Minutolo 1 Daniela Alioto 1
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Francesco Di Serio 2
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Maria Cinque 1 & Pasquale Lombardi 1,3 & Beatriz Navarro 2
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Received: 3 March 2020 / Accepted: 20 March 2020 # Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2020
Abstract Citrus concave-gum associated virus (CCGaV) and citrus virus A (CiVA) are two negative-stranded RNA viruses infecting citrus. Both viruses have been identified by high-throughput sequencing in Italy and their simultaneous presence in the same host documented. While CCGaV has been associated with citrus concave gum disease, the association of CiVA with symptoms is still unclear. Since the two virus genomes share about 75% sequence identity, their identification by RT-PCR is not obvious. Here, a multiplex RT-PCR to simultaneously detect and identify them is reported. Amplification of the citrus cytochrome oxidase (COX) gene transcript was included as internal control to minimize the risk of false negatives. Specificity and sensitivity of multiplex RTPCR were comparable to that of conventional uniplex RT-PCR set up in previous studies for the independent detection of the two viruses. The method was validated by a field survey performed testing almost 300 trees grown in different areas of southern Italy. The assay revealed infection by CCGaV and CiVA in 17.7% and 18.4% of the tested trees, respectively, with mixed infection recorded in 5.0% of the samples. The novel mRT-PCR method saves time and costs and has the potential to be used for largescale surveys and certification programs. Keywords Citrus viruses . Detection . Multiplex RT-PCR assay . Internal control, cox gene transcripts
Citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) have been recently identified in citrus trees by highthroughput sequencing (HTS) (Navarro et al. 2018a, b). CCGaV has been classified as the reference species (Citrus coguvirus) of the new genus Coguvirus within the order Bunyavirales (Abudurexiti et al. 2019). Due to the close relationship with CCGaV, CiVA has been proposed as a second species in the same genus (Navarro et al. 2018b). In southern Italy, where the virus was first identified, CCGaV has been
* Beatriz Navarro [email protected] * Daniela Alioto [email protected] 1
Dipartimento di Agraria, Università degli Studi di Napoli Federico II, Portici, NA, Italy
2
Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Bari, Italy
3
CREA Research Centre for Vegetable and Ornamental Crops, Pontecagnano-Faiano, SA, Italy
associated with concave gum-blind pocket (CG), a severe and widespread citrus disease, first described in 1930s in California (Fawcett 1936). Interestingly, CCGaV has been recently identified in apple trees showing bark symptoms in Washington State (Wright et al. 2018), indicating that it may have a wide host range and distribution. CiVA, also reported in southern Italy for the fir
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