MOLECULAR STUDIES ON VIRUSES ASSOCIATED WITH DISEASES OF TWO SMALL FRUIT CROPS IN WASHINGTON STATE

Arunabha Mitra

doi:10.7273/000003362

This research was focused on economically important viruses infecting wine grapes (Vitis vinifera) and highbush blueberry (Vaccinium corymbosum L.). In the first objective, genetic diversity of Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus, family Closteroviridae) in Washington vineyards was examined. Total RNA preparations from GLRaV-3-positive samples, collected between 2015 and 2019 from red- and white-fruited wine grape cultivars from commercial vineyards in four American Viticultural Areas, were subjected to Illumina© sequencing to generate near-complete viral genome sequences. A global phylogenetic analysis of these sequences revealed the presence of diverse genetic variants of GLRaV-3 as single and mixed infections than previously reported from other grapevine-growing regions. In the second objective, minireplicon cDNA clones were built for WA-CH variant of GLRaV-1 as a simplified reverse genetics system. The functionality of minireplicon cDNA clones, containing the 5ʹ-non-translated region (NTR)-replication gene block-3ʹ-NTR cassette with a green fluorescent protein (GFP) reporter gene, was confirmed by the expression of GFP in Nicotiana benthamiana leaves agro-coinfiltrated with heterologous silencing suppressors. Mutational analysis showed that the first 32 nucleotides at the 5ʹ-terminus of the 5ʹ-NTR, highly conserved among GLRaV-1 genetic variants, are sufficient for RNA replication. Further studies showed that 5ʹ-NTR sequences from distinct variants of GLRaV-1 are compatible with the replicase gene function of the WA-CH variant. In contrast, the GLRaV-1 minireplicon was non-functional when its 5ʹ-NTR was replaced with corresponding sequences from other GLRaV genotypes. In the third objective, RNA1 and RNA2 genome segments of a Tobacco ringspot virus (TRSV, genus Nepovirus, family Secoviridae) isolate were molecularly characterized from highbush blueberry plants showing severe defoliation, stunting, and decline symptoms. Symptomatic plants produced small, uneven-sized berries with delayed ripening and poor quality compared to berries produced by healthy plants. In bait plant assays, nematode transmission of TRSV was demonstrated from virus-infected blueberry plants to healthy cucumber plants. Using morphological characteristics and sequences corresponding to the D2-D3 expansion segment and ITS rRNA-coding regions, Xiphinema rivesi was identified as the dagger nematode in soil samples collected near symptomatic blueberry plants. Detection of TRSV in nematodes implicated X. rivesi as a putative vector of the virus.

MOLECULAR STUDIES ON VIRUSES ASSOCIATED WITH DISEASES OF TWO SMALL FRUIT CROPS IN WASHINGTON STATE

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