First Report of Grapevine Leafroll-Associated Virus-3 Infecting <i>Vitis vinifera</i> Grape in Alabama

Grapevine leafroll-associated virus-3 (GLRaV-3) is considered the most predominant cause of grapevine leafroll disease (GLD), one of the most destructive viral diseases affecting grapevines and wine production worldwide (Maree et al. 2013). GLRaV-3 is a positive-sense, single-stranded RNA (+ssRNA) virus in the family Closteroviridae, genus Ampelovirus, species tritis (Martelli et al., 2012; Maree et al., 2013). Grape plants (cultivar ‘Errante Noir’) were planted at the Chilton Research and Extension Center (CREC), Chilton County, Alabama, in the spring of 2024. In November 2024, a plant exhibited symptoms of dark-purplish red veins with cupping of the leaf margins, and in July 2025, symptoms of reddish-brown blotches randomly distributed on the foliage appeared (Supplemental Figure 1: A: I-II). To investigate if a pathogen caused this, a sample was sent to Agdia, Inc. (Elkhart, IN, USA), where tissues were subjected to a pathogen screen, including alfalfa mosaic virus (AMV), arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV), Phytophthora (Phyt), peach rosette mosaic virus (PRMV), strawberry latent ringspot virus (SLRSV), tomato ringspot virus (ToRSV), tobacco ringspot virus (TRSV), GLRaV-3, grapevine pinot gris virus (GPGV), grapevine red blotch-associated virus (GRBaV), and Xylella fastidiosa (Xf). The sample tested negative for all except for GLRaV-3. To confirm GLRaV-3, RT-PCR was used. Total RNA was extracted from 0.1 g petiole tissue from three subsamples using the RNeasy Plant Mini Kit (Qiagen), producing three separate RNA samples. The cDNA was synthesized using SuperScript IV Reverse Transcriptase (Invitrogen), and Platinum Taq DNA Polymerase (Invitrogen) was used to amplify the heat shock protein (HSP70) and the coat protein (CP) using specific primers (Thompson et al. 2019). PCR products were checked on the TapeStation using the D1000 ScreenTape kit (Agilent), and amplicons corresponding to 600 bp (HSP70) and 280 bp (CP) were detected (Supplemental Figure 1: B). Sanger sequencing was conducted at Azenta Life Sciences in both directions. The manufacturer’s instructions were followed in all used kits. Sequences were analyzed using BLASTn (Altschul et al. 1990) to confirm that all three sequences from each gene match GLRaV-3. Sequences were submitted to GenBank (accessions: PX123059-64), and an isolate name was given (GLRaV-3_Chilton-AL.1.1-3). The closest BLAST hit is GLRav3-8415B (KY073324.1) with 100% (HSP70), 98.24% (CP) nucleotide similarities (Supplemental Table 1). The CP sequences of GLRaV-3_Chilton-AL.1.1-3 were compared to 139 isolates of GLRaV-3 from GenBank (Supplemental Table 1) to construct a maximum likelihood tree using methods described by Shehata et al. (2025), with a modification where IQ-TREE was used to construct the tree with 1000 replicates (Nguyen et al. 2015). This tree indicated that GLRaV-3_Chilton-AL.1.1-3 is within group XII, with two isolates from Canada (Supplemental Figure 1: C). Other grape plants planted along with the infected plant at the Chilton Co. site (n=15) were also tested for GLRaV-3 as described above, and all tested negative. This constitutes the first report of GLRaV-3 in Alabama, highlighting the importance of purchasing clean material to prevent introduction of the disease in vineyards, a crop with a total impact of $1.5 billion in Alabama (Good, T. 2023).