Identification and characterization of Citrus yellow vein clearing virus, a putative new member of the genus Mandarivirus infecting Citrus spp.

Authors: LOCONSOLE, G. - University Of Bari; ONELGE, N - Cukurova University; POTERE, O - University Of Bari; GIAMPETRUZZI, A - University Of Bari; BOZAN, O - University Of Cukurova; SATAR, S - University Of Cukurova; DE STRADIS, A - National Research Council - Italy; SAVINO, V - University Of Bari; Yokomi, Raymond - Ray; SAPONARI, M - National Research Council - Italy

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/14/2012
Publication Date: 8/22/2012
Citation: Loconsole, G., Onelge, N., Potere, O., Giampetruzzi, A., Bozan, O., Satar, S., De Stradis, A., Savino, V., Yokomi, R.K., Saponari, M. 2012. Identification and characterization of Citrus yellow vein clearing virus, a putative new member of the genus Mandarivirus infecting Citrus spp. Phytopathology. 102: 1168-1175.

Interpretive Summary: A few lemon and sour orange trees showing symptoms of strong yellow vein clearing and leaf distortion were observed in 2000 on the experimental farm of 'ukurova University, Adana, Turkey. The pathogen was aphid transmissible and spread rapidly to other citrus trees. Therefore, the disease is invasive and is a threat to Turkey and other citrus-producing countries. From previous reports from Pakistan and India, the disease was provisionally named Citrus yellow vein clearing virus (CYVCV) and was associated with filamentous virions and was graft transmissible to citrus and mechanically transmissible to herbaceous indicator plants. CYVCV shares many attributes with Indian citrus ringspot virus (ICRSV) (genus Mandarivirus). However, as the names implies, the two diseases have different symptomologies on citrus. To characterize CYVCV, the strain Y1 from Adana, Turkey, was purified from infected citrus and beans and a polyclonal antiserum was produced that reacted with CYVCV-Y1 but not ICRSV. Electron microscopy of symptomatic tissue using gold-labeled antiserum, confirmed that the observed flexuous filamentous virus-like particles were CYVCV. The whole virus genome of CYVCV-Y1 was reconstructed from sequenced small interfering RNA fractions isolated from graft-inoculated lemon plants. The genome encoded six open reading frames (presumptive genes) and had a positive-sense RNA of 7,529 nucleotides in size. Genome structure was typical for the family Alphaflexiviridae , but sequence identity with ICRSV was only ~74%. Phylogenetic analysis showed CYVCV and ICRSV were different species but on the same branch clearly apart from other viruses in the Alpha- and Betaflexiviridae families. Primers were designed based on the presumptive CYVCV coat protein gene which successfully detected the virus by conventional and quantitative reverse transcription polymerase chain reaction (PCR) assays in infected plants. The assays detected CYVCV from plants experimentally infected with the Y1 strain as well as from symptomatic citrus trees from Turkey. No PCR products developed from ICRSV, healthy controls, or a large series of citrus viruses and viroids. These data suggest that CYVCV-Y1 characterized in this study is the causal agent of CYVCV. Furthermore, the data supports the hypothesis that CYVCV represents a new species in the genus Mandarivirus.

Technical Abstract: Yellow vein clearing virus, an uncharacterized filamentous virus, was first observed in Pakistan in 1988 and later in India in 1997 in Etrog citron (Citrus medica). Based on electron microscopic evidence of filamentous particles, the virus, provisionally named Citrus yellow vein clearing virus (CYVCV), was purified and a specific polyclonal antisera was developed, indicating that CYVCV was serologically distinct from other filamentous viruses. In 2000, a virus, similar to that reported in Pakistan and India, was found in Turkey and was associated with lemon (Citrus limon L.) and sour orange (C. aurantium). Electron microscopy from symptomatic lemon and sour orange trees confirmed presence of filamentous viral particles. CYVCV isolate Y1 from Adana, Turkey, was used in greenhouse indexing tests to mechanically transmit the virus to Chenopodium quinoa, C. amaranticolor and Phaseolus vulgaris as well as graft-transmission to Eureka lemon. Symptoms in indicator hosts were identical to those described in previous reports of yellow vein clearing disease in Pakistan and India. CYVCV was shown to share the same particle morphology and mechanical transmission host range as that reported for the Indian citrus ringspot virus (ICRSV) (family Alphaflexiviridae, genus Mandarivirus). To examine the putative genome of CYVCV isolate Y1, small RNA fractions from graft-inoculated and symptomatic Eureka lemon were isolated and sequenced using the HiSeq2000 platform and the whole genome of the putative virus reconstructed. The genome sequence indicated that it encodes six open reading frames and has a positive-sense RNA of 7,529 nucleotides in size. Sequence comparison with ICRSV, however, indicated only ~74% overall nucleotide sequence identity. Although CYVCV was similar to ICRSV with regard to its genome organization, viral particles and herbaceous host range, the two pathogens were biologically, serologically and molecularly distinct and cause different diseases. CYVCV was found mainly in lemon varieties causing a strong yellow vein clearing and leaf distortion; whereas ICRSV was found primarily in mandarin causing chlorotic rings on leaves which drop prematurely. These data suggest that CYVCV is an appropriate name for the causal agent of disease and a new species designation for the virus is proposed in the genus Mandarivirus.