Characterization of Four Viral Species Belonging to the Family Potyviridae Isolated from Ranunculus asiaticus

Abstract

Four different viral species were isolated from diseased Ranunculus asiaticus plants growing in Imperia Province (Italian Riviera-Liguria Region). Infected plants exhibited mosaic symptoms and growth abnormalities. The viruses were mechanically inoculated to a range of herbaceous hosts and differentiated biologically. Long flexuous particles were present in leaf dip extracts observed by electron microscopy. A general protocol for the amplification of potyvirus genome fragments through reverse transcription-polymerase chain reaction generated products that were cloned and sequenced. Sequence and phylogenetic analysis suggested that three of these isolates could be considered new viral species belonging to the genus Potyvirus. The fourth isolate is a new member of the genus Macluravirus. Purified virus was used as antigen to produce a specific polyclonal antiserum in rabbit; serological features were established through double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), antigen coated plate (ACP)-ELISA, and western blot analysis. DAS-ELISA was highly specific for each virus isolate, whereas some cross-reactivity was shown in ACP-ELISA and western blot analysis. Aphid transmission by Myzus persicae was demonstrated in a controlled environment for each of the four viral isolates, whereas no transmission through seed was observed.

RT‐PCR tests for sensitive detection of the major Ranunculus‐infecting viruses: field and in vitro applications

“…Double antibody sandwich (DAS)‐ or triple antibody sandwich (TAS)‐ELISA were performed on ranunculus samples using 13 virus‐specific ELISA kits (for DAS‐ELISA, coating: purified immunoglobulins G (IgG) from specific antiserum, and conjugate: alkaline phosphatase‐conjugated specific IgGs). Specific antisera were obtained by rabbit immunization according to standard procedures (Turina et al ., ) using suspensions of virus particles of the different isolates, purified from infected plant tissues, according to virus particle shape and physical/chemical characteristics. The ELISA kits, validated at IPSP‐CNR, Torino, Italy are currently used for routine virus detection; homologous antigen detection limits were calculated for each ELISA kit using serial dilutions (1:10 to 1:50 000) of infected plant sap in extraction buffer (phosphate‐buffered saline, pH 7.4 (PBS), 0.05% Tween, 2% PVP) in order to check all kit performances and establish optimal antigen and antibody dilutions for each virus system (Table ).…”

“…Ranunculus hybrids, subsequently referred to as ranunculus, are susceptible to infection by several viruses, in most cases found in mixed infection, all associated with economically important diseases (Vaira et al ., ; Garibaldi et al ., ; Turina et al ., ; Ciuffo et al ., ; Restuccia et al ., ). Common viral symptoms on ranunculus are mottling, chlorosis, mosaic, necrosis and deformation on leaves, vein yellowing, stunting of the plant and deformation and colour breaking on flowers.…”

A New Virulent Isolate of Clover Yellow Vein Virus on Phaseolus vulgaris in Bulgaria

“…White colonies where screened for presence of the insert with colony‐PCR using the universal primers M13F and M13Rev as previously described (Turina et al. ). Plasmids from positive colonies were extracted with Zyppy™ Plasmid Miniprep Kit (Zymo Research Orange, CA, USA) and sequenced.…”

A New Tospovirus sp. in Cucurbit Crops in Mexico

“…Leaves of symptomatic C. melo and N. benthamiana plants showing strong mosaic symptoms 2 weeks post inoculation were homogenized with 50 mM phosphate buffer, pH 7, containing 1 mM Na-EDTA, 5 mM Na-DIECA, and 5 mM Na-thioglycolate (extraction buffer) and mechanically inoculated onto leaves of a series of herbaceous plants to determine the partial host range (Table 1) as previously described (35). Leaves of symptomatic C. melo and N. benthamiana plants showing strong mosaic symptoms 2 weeks post inoculation were homogenized with 50 mM phosphate buffer, pH 7, containing 1 mM Na-EDTA, 5 mM Na-DIECA, and 5 mM Na-thioglycolate (extraction buffer) and mechanically inoculated onto leaves of a series of herbaceous plants to determine the partial host range (Table 1) as previously described (35).…”

“…The specificity of purified MeSMV-IgG was assessed by antigen-coated plate (ACP) ELISA using dilutions of infected and healthy plants (35). The specificity of purified MeSMV-IgG was assessed by antigen-coated plate (ACP) ELISA using dilutions of infected and healthy plants (35).…”