First Report of Apple Stem Grooving Virus in Apple and Pear Trees in Azerbaijan

Pome fruits from the family Rosaceae are grown globally. Viruses can affect their quality, shape, and size. Apple stem grooving virus (ASGV, genus Capillovirus, family Betaflexiviridae) has a wide host range (Chofong et al., 2024), including apple and pear trees (Mustafayev, 2021). No information is available on the occurrence of ASGV in Azerbaijan. The aim of this research was to survey apple, pear and quince in commercial orchards and private gardens of various cultivars in different geographical areas of Azerbaijan for ASGV. A total 87 leaf samples was collected in 2017-2019 from 38 apple (Malus domestica L.), 27 pear (Pyrus communis L.), and 22 quince (Cydonia oblonga L.) trees exhibiting stem deformation and foliar necrotic spots for DAS-ELISA testing using ASGV serological reagents (Neogen - Scotland, UK). Positive reactions to ASGV were obtained for 27 apple, 16 pear, and 6 quince samples. To confirm the presence of ASGV, total RNA was isolated from 67 leaf samples (0.5 g), as previously described (Foissac et al., 2005), and analyzed by RT-PCR using the OneStep RT-PCR kit (Qiagen) and primers designed in the coat protein gene of ASGV (F: 5'-GCCACTTCTAGGCAGAACTCTTTGAA-3' and R: 5'-AAGCATGACTTCCTGTTTTTCCCCAA-3') and the NADH dehydrogenase subunit 5 fragment used as a housekeeping gene (Menzel et al., 2002). RT-PCR analyses were performed for 5 min at 95 °C and 34 cycles of 10 s at 95 °C, 20 s at 50 °C, 20 s at 72 °C, followed by a final extension of 2 min at 72 °C. DNA amplicons were resolved by electrophoresis on 2% agarose gels and visualized under UV illumination after staining with GelRed (Biotium, Fremont, CA) (Mustafayev et al., 2025). Results showed ASGV DNA amplicons of the expected size (273 bp) in 17 apple and 2 pear samples but not in quince samples. Discrepancies between DAS-ELISA and RT-PCR results are unclear but could be due to the primers not optimally capturing the ASGV genetic variability. DNA amplicons corresponding to one pear (PV098407) and five apple (PV098408-PV098412) samples were cleaned with Exo SAP-IT and characterized by bidirectional Sanger sequencing at the Cornell Biotechnology Resource Center in Ithaca, NY. Sequence analyses with the DNASTAR Lasergene software package (v17.3.0.57) showed a high sequence identity ranging from 98.2% to 98.9% and 97.8% to 100% at the nucleotide and amino acid, respectively, among Azerbaijani ASGV isolates and other apple and pear isolates from South Korea (JN792474, JN792477), the USA (MW322834), Pakistan (MN861413), China (OM313348), Germany (MW582790), and Brazil (MK929791). Azerbaijani ASGV isolates Aghdam 81 and Xachmaz 256 were co-infected ACLSV, isolate Sheki 27 with apple chlorotic leaf spot virus, and isolates Guba 101 and Shemkir 174 with apple stem pitting virus, as shown by RT-PCR. In consistence with other reports, stem and leaf symptoms were not associated with the occurrence of ASGV in single or mixed infections, and sequence identities of the partial ASGV coat protein gene were not distinct between apple and pear isolates (Shokri et al., 2023). This is the first report of ASGV in apple and pear trees in Azerbaijan.