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Abstract:
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At least 58 viruses have been reported to infect grapevines causing economic damage globally. Conventional
detection strategies based on serological assays, biological indexing and RT-PCR targeting one or
few viruses in each assay are widely used. Grapevines are prone to contain mixed infections of several
viruses, making the use of these techniques time-consuming. A 70-mer oligonucleotide microarray able
to detect simultaneously a broad spectrum of known viruses as well asnewviruses by cross-hybridization
to highly conserved probes is reported in the present study. The array contains 570 unique probes
designed against highly conserved and species-specific regions of 44 plant viral genomes. In addition
probes designed against plant housekeeping genes are also included. By using a random primed RT-PCR
amplification strategy of grapevine double stranded RNA-enriched samples, viral agents were detected
in single and mixed infections. The microarray accuracy to detect 10 grapevine viruses was compared
with RT-PCR yielding consistent results. For this purpose, grapevine samples containing single or mixed
infections of Grapevine leafroll-associated virus-1, -2, -3, -4, -7, -9, Grapevine fanleaf virus, Grapevine
rupestris stem pitting-associated virus, Grapevine virus A, and Grapevine virus B were used. Genomic
libraries containing complete viral genomes were also used as part of the validation process. The specific
probe hybridization pattern obtained from each virus makes this approach a powerful tool for high
throughput plant certification purposes and also for virus discovery if the new viral genomic sequences
have partial similarity with the microarray probes. Three Closteroviridae members (Grapevine leafrollassociated
virus -4, -7 and -9) were detected for the first time in Chilean grapevines using the microarray. |