Alei v2

To determine the phylogenetic clade of GRBV isolates in symptomatic, infected grapevines, PCR products corresponding to a fragment of the GRBV replication ORF [22 (link),36 (link),38 (link)] were Sanger sequenced at the Cornell Biotechnology Resource Center in Ithaca, New York. GRBV sequences were assembled and analyzed using DNASTAR Lasergene software suite, version 14.1. Additionally, some PCR products underwent restriction digestion with AleI-v2 (New England Biolabs, Ipswich, MA, USA) to determine the GRBV phylogenetic clade, as previously described [41 (link)]. Digestions were resolved via electrophoresis on agarose gels and visualized using UV illumination post-staining with GelRed (Biotium, Fremont, CA, USA). Restriction digests of approximately 201 and 117 bp in size reflected a GRBV clade 1 isolate, while a single uncut 318 bp fragment indicated a GRBV clade 2 isolate [41 (link)].

To determine the phylogenetic clade of GRBV isolates in plant samples and S. festinus specimens, PCR products amplifying a fragment of the GRBV replication ORF [3 (link),15 (link),37 (link)] from plant and insect nucleic acids were restriction digested with AleI-v2 (New England Biolabs, Ipswich, MA, USA) at 37 °C for two hours. Each reaction contained 5 µL of PCR reaction and 2 µL of rCutSmart™ buffer (New England Biolabs), for a final volume of 20 µL. Digestions were resolved by electrophoresis on agarose gels and visualized using UV illumination post-staining with GelRed (Biotium, Fremont, CA, USA). In addition, PCR products were Sanger sequenced at the Cornell Biotechnology Resource Center in Ithaca, New York, to determine the accuracy of the restriction digests in distinguishing GRBV isolates from distinct phylogenetic clades. GRBV sequences were assembled using the DNASTAR Lasergene software suite, version 14.1.