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ISHS Acta Horticulturae 899: International Symposium on Plum Pox Virus

DETECTION OF A NEW WINONA-LIKE PLUM POX VIRUS ISOLATE IN NATURALLY INFECTED CANADIAN PLUM (PRUNUS NIGRA) IN RUSSIA

Authors:   A. Sheveleva, S. Chirkov, E. Nemova
Keywords:   strain typing, W strain, coat protein, Western blot, IC-RT-PCR, genome sequence
Abstract:
A new Winona-like isolate of Plum pox virus (PPV) designated as 1410 has been found in naturally infected Canadian plum (Prunus nigra Ait.) plants introduced in the Botanical Garden (Moscow, Russia). The 1410 isolate was detected in infected leaves of adult seed-borne trees by double antibody sandwich (DAS)-ELISA using the PPV Reagent Set SRA 31505 (Agdia), indirect DAS-ELISA using a PPV-Universal ELISA kit (Agritest, Italy), and by immunocapture reverse-transcription polymerase chain reaction (IC-RT-PCR) performed with universal P1/P2 and 3’NCR-specific primers. In IC-RT-PCR analysis with W-specific primers the 1410 isolate gave a 327-bp PCR product similar to that of the W3174 isolate, a representative member of the Winona group. No amplification with D, M, C, or Rec strain-specific primers and reaction with PPV-D, M, or C specific monoclonal antibodies in DASI-ELISA was observed. Like W3174, the 243-bp PCR fragment of the 1410 isolate obtained with P1/P2 primers was resistant to the cleavage by AluI and RsaI endonucleases. The (Cter)NIb-CP-3’NCR nucleotide sequence of the 1410 isolate was determined. The coat protein (CP) sequence analysis showed that the 1410 isolate was closely related to W3174 (James and Varga, 2005) and 44191 (Mavrodieva et al., 2006) isolates comprising the Winona group. Analysis of deduced amino acid CP sequences revealed significant differences within the end of N-terminus of Winona CPs. The 1410 isolate was mechanically sap-transmitted to Nicotiana benthamiana. Systemic infection in N. benthamiana plants was symptomless and the virus titer was moderate. Molecular weight of the 1410 isolate CP determined by Western blotting analysis of P. nigra and N. benthamiana extracts was about 42 kDa apparently due to abnormally slow CP mobility.
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