Detection and typing of prunus viruses in plant tissues and in vectors by print and spot-capture PCR, heminested-PCR and PCR-ELISA

Abstract

A very sensitive and simple procedure has been developed for the preparation of plant and insect vector samples before PCR amplification using direct tissue printing or squashing on paper. The print-capture PCR (PC-PCR) technique was successfully used for the detection of plum pox potyvirus (PPV), apple chlorotic leaf spot trichovirus (ACLSV) and prunus necrotic ring spot ilarvirus(PNRSV) and apple mosaic (ApMV) ilarvirus, in imprints of different stone fruit species, avoiding preparation of extracts. The immobilized targets in imprinted paper can be stored and/or mailed before PCR amplification. The system was used for the simultaneous detection and typing of PPV isolates belonging to the M or D serotypes, in plant tissues and in individual Aphis gossypii vectors, by two new methods: heminested print or spot-capture PCR (H-PCR, H-SC-PCR) and PCR-ELISA (post-PCR-hybridization-ELISA detection of digoxigenin-label amplicons according to Boehringer Mannheim). An excellent coincidence of typing PPV isolates was obtained by ELISA-DASI (using specific monoclonal antibodies to PPV-D or M) or by nucleic acids-based techniques. ELISA-DASI with monoclonal antibodies, continues to be a suitable technique for the routine detection of PPV in apricot field samples in spring. Nevertheless, the IC-PCR appears to be more powerful in summer and winter. The simultaneous detection and typing of PPV targets in individuals of A.gossypii by H-SC-PCR demonstrates the feasibility of detecting nonpersistently transmitted viruses in aphid vectors previously squashed on paper and the possibility of segregation of subisolates from a mixture of D and M isolates of PPV.