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Detection of apple chlorotic leaf spot virus and apple stem grooving virus by RT-PCR and IC-RT-PCR



Detection of apple chlorotic leaf spot virus and apple stem grooving virus by RT-PCR and IC-RT-PCR



Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen Universiteit Gent 60(2A): 277-287



Apple chlorotic leafspot virus (ACLSV) and Apple stem grooving (ASGV) isolates, and complexes containing these viruses have been transferred and multiplied on herbaceous host plants in the greenhouse for the development of RT-PCR and IC-RT-PCR detection methods. Comparative analysis of nucleotide and deduced amino acid sequences published for 2 isolates of ACLSV and one of ASGV showed the presence of several short stretches of homologous amino acids although both viruses belong to different phytovirus genera. Only amino acid sequence homologies located in the 3' terminal part of the RNA polymer= gene are sufficiently conserved and have a sufficient length to allow the design of different sets of degenerate primers. These primers have been used for amplification of viral sequences from reverse transcribed total RNA preparations of virus-infected leaves of Chenopodium quinoa or Nicotiana occidentalis. The primers pairs retained have been also tested for immunocapture RT-PCR amplification of genomic RNA from virus particles contained in crude sap of the same plant species. As the size of amplified products are quite similar for ACLSV and ASGV, the identification of the responsible virus would require the combination of PCR and hybridisation with specific probes from cloned cDNA. The RT-PCR and IC-RT-PCR protocols thus developed will be adapted for direct use on woody material, and specially mother tree with the aim to define certification scheme for virus free propagating material.

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