Simultaneous detection of citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) by multiplex RT-PCR

Minutolo, Maria; Serio, Francesco Di; Cinque, Maria; Lombardi, Pasquale; Navarro, Beatriz; Alioto, D.

doi:10.1007/s42161-020-00542-1

Cited by 11 publications

(8 citation statements)

References 27 publications

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“…In general, the NP gene of a virus is usually considered an appropriate target for its detection because of its high expression. Recently a multiplex RT-PCR protocol was proposed for the simultaneous detection of CiVA and CCGaV targeting the NP gene of the two viruses (Minutolo et al 2020). However, in the present study the NP targeting RT-(q)PCRs produced a number of false negative results.…”

Section: Discussionmentioning

confidence: 59%

Association of Citrus Virus A to Citrus Impietratura Disease Symptoms

et al. 2021

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Citrus impietratura disease (CID) is a graft transmissible, virus-like disease observed in old-line citrus trees; its characteristic symptom is the appearance of gum in the albedo of the affected fruits. To identify the causal agent of the disease, high throughput sequencing (HTS) was performed on symptomatic orange fruits. The analysis of the obtained data revealed in all samples, mixed infections of viroids commonly found in citrus trees together with the recently described citrus virus A (CiVA). Examination of additional symptomatic fruits with conventional RT-PCRs led to the identification of a single CiVA infection in one tree, which was verified by HTS. Indexing of the single CiVA-infected tree on indicator plants resulted in the appearance of characteristic symptoms in the leaves that were correlated with virus accumulation. Moreover, a comparative analysis among symptomatic and asymptomatic fruits derived from the same trees was performed and included the single CiVA-infected orange tree. The analysis revealed a positive correlation between the appearance of symptoms and the accumulation of CiVA RNAs. To facilitate CiVA detection during certification programs of propagation material a quantitative RT-qPCR targeting the movement protein of the virus, was developed and evaluated for reliable and sensitive detection of the virus. To the best of our knowledge this is the first study that associates CiVA with the appearance of impietratura disease symptoms.

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Section: Discussionmentioning

confidence: 59%

Association of Citrus Virus A to Citrus Impietratura Disease Symptoms

et al. 2021

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“…A rabbit polyclonal antiserum (CCGaV-ab1) against the N-terminal region (amino acids 1 to 119) of the CCGaV NP was generated and used to develop a DAS-ELISA protocol, in collaboration with Agritest. In selecting the protein region to be used to develop an antiserum specific for CCGaV, the possibility of cross-reaction of polyclonal antibodies with the NP of the closely related coguvirus CiVA, also infecting citrus [13], was taken into consideration. Indeed, the amino acid identity between the N-terminal region (amino acids 1 to 119) of the NP of the two viruses was only 37.29%, much lower than the identity in the rest of the protein (73.16%).…”

Section: Ccgav Is Specifically Detected By Standard Double-antibody Sandwich Enzyme-linked Immunosorbent Assay (Das-elisa) Using a Polyclmentioning

confidence: 99%

“…Several citrus host species previously tested for CCGaV and CiVA [13] were selected for this preliminary step. Reliability of the DAS-ELISA protocol was first tested by comparing the DAS-ELISA with the RT-PCR assay based on the primers CG.15 and CG.20 (Table S1).…”

Section: Ccgav Is Specifically Detected By Standard Double-antibody Sandwich Enzyme-linked Immunosorbent Assay (Das-elisa) Using a Polyclmentioning

confidence: 99%

“…Total RNA was extracted from 200 mg of leaf tissues and reverse transcribed as reported previously by Minutolo et al (2020). PCR amplification was carried out in 25 µL of reaction mixture using 1µL of cDNA, 1 U GoTaq Hot Start Polymerase (Promega, Madison, WI, USA) and specific primers for each tested virus at a final concentration of 0.2 µM (Table S1).…”

Section: Rna Extraction and Rt-pcrmentioning

confidence: 99%

“…Molecular methods based on RT-PCR to specifically detect CCGaV and CiVA have been developed since early reports of these viruses [1,8]. Moreover, a multiplex RT-PCR protocol to identify and discriminate in a single reaction the two viruses in citrus has also been fixed recently [13], and an RT-qPCR has been applied for quantitative assays of CiVA in citrus fruits [9] and for the detection of CCGaV, CiVA and other negative-stranded RNA viruses [14]. Although these molecular methods allow detection of coguviruses, the availability of serological assays would strongly reduce the detection costs and provide new opportunity for large-scale surveys and for extensive tests requested by certification programs of vegetative propagation materials.…”

Section: Introductionmentioning

confidence: 99%

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Identification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches

Minutolo

Cinque

Chiumenti

et al. 2021

Plants

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Citrus concave gum-associated virus (CCGaV) is a negative-stranded RNA virus, first reported a few years ago in citrus trees from Italy. It has been reported in apple trees in the USA and in Brazil, suggesting a wider host range and geographic distribution. Here, an anti-CCGaV polyclonal antiserum to specifically detect the virus has been developed and used in a standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that has been validated as a sensitive and reliable method to detect this virus both in citrus and apple trees. In contrast, when the same antiserum was used in direct tissue-blot immunoassay, CCGaV was efficiently detected in citrus but not in apple. Using this antiserum, the first apple trees infected by CCGaV were identified in Italy and the presence of CCGaV in several apple cultivars in southern Italy was confirmed by field surveys. High-throughput sequencing (HTS) allowed for the assembling of the complete genome of one CCGaV Italian apple isolate (CE-c3). Phylogenetic analysis of Italian CCGaV isolates from apple and citrus and those available in the database showed close relationships between the isolates from the same genus (Citrus or Malus), regardless their geographical origin. This finding was further confirmed by the identification of amino acid signatures specific of isolates infecting citrus or apple hosts. Analysis of HTS reads also revealed that the CE-c3 Italian apple tree, besides CCGaV, was simultaneously infected by several viruses and one viroid, including apple rubbery wood virus 2 which is reported for the first time in Italy. The complete or almost complete genomic sequences of the coinfecting agents were determined.

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Duplex PCR assay for detection of chickpea chlorotic dwarf virus and peanut witches’ broom phytoplasma in chickpea

Reddy

Rao

2021

3 Biotech

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Simultaneous detection of citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) by multiplex RT-PCR

Cited by 11 publications

References 27 publications

Association of Citrus Virus A to Citrus Impietratura Disease Symptoms

Association of Citrus Virus A to Citrus Impietratura Disease Symptoms

Identification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches

Duplex PCR assay for detection of chickpea chlorotic dwarf virus and peanut witches’ broom phytoplasma in chickpea

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