Characterization of Tobacco Ringspot Virus and Its Nematode Vector, Xiphinema rivesi, Infecting Highbush Blueberry in...

Highbush blueberry (Vaccinium corymbosum L.) plants showing stunting and severe defoliation were observed in an organic blueberry farm in eastern Washington state. Initially, leaves of affected blueberry plants showed crinkling and mottling, chlorotic spots, and red-colored rings with chlorotic centers. Subsequently, infected plants showed severe defoliation with overall decline within a few years. Symptomatic blueberry plants produced small berries of uneven size and poor ripening compared with berries of uniform ripening produced by healthy plants. Berries from symptomatic plants showed significant reduction in total soluble solids, juice pH, and total anthocyanins and increased titratable acidity compared with berries from healthy plants. High-throughput sequencing of total RNA extracted from symptomatic leaves revealed the presence of tobacco ringspot virus (TRSV; genus Nepovirus, family Secoviridae). The complete genome sequence of the RNA1 and RNA2 segments was determined, respectively, to be 7,512 and 3,925 nucleotides, excluding the 3'-terminal poly(A) tail. In reverse transcription PCR assays, a portion of the Pro-Pol domain of RNA1 and the coat protein (CP) gene encoded by RNA2 were amplified only from symptomatic blueberry plants. In phylogenetic analyses, the TRSV isolate from blueberry clustered with viral species in Nepovirus subgroup A based on the conserved CG-GDD motif of the Pro-Pol domain of RNA1 and the conserved sequence in the CP of RNA2. The detection of TRSV in adult female dagger nematodes extracted from soil samples collected near symptomatic blueberry plants and in-field transmission assays using cucumber bait plants implicated nematode transmission of TRSV. The dagger nematode was identified as Xiphinema rivesi Dalmasso, 1969 using a combination of morphological and morphometric data and phylogenetic analysis of nucleotide sequences specific to D2-D3 expansion domains of the 28S rRNA gene and the internal transcribed spacer region of nematode species within the genus Xiphinema.