First Report of Apple Stem Pitting Virus in Pome Fruit Trees in Azerbaijan

Pome fruits (family Rosaceae) are extensively produced throughout Azerbaijan. Apple stem pitting virus (ASPV, Foveavirus mali, genus Foveavirus, family Betaflexiviridae) has a host range that includes pome and stone fruit trees (Mustafayev 2021). No information is available on the occurrence of ASPV in Azerbaijan. The aim of this research was to survey Azerbaijani pome fruit orchards for ASPV. A total of 67 leaf samples were collected from 27 pear (Pyrus communis L.), 38 apple (Malus domestica Borkh.) and 2 quince (Cydonia oblonga Mill.) trees in 2018-2019. Twenty apple trees and 13 pear trees displayed veinal yellowing foliar symptoms, and the two quince trees showed deformation. Leaf samples were analyzed by DAS-ELISA using ASPV-specific serological reagents (Neogen, Ayr, Scotland, UK). ASPV was detected in three pear and five apple samples but not in quince samples. To confirm the presence of ASPV in the two pome fruit tree hosts, total RNA was isolated from symptomatic leaves (0.5 g) from trees that tested positive in DAS-ELISA, as previously described (Foissac et al. 2005), and analyzed by multiplex RT-PCR using the OneStep RT-PCR kit (Qiagen) and primers designed in the coat protein gene of ASPV (F: 5′-GCAGCTTTTGACTTCTTCTTTGGAG-3′ and R: 5’-CTGATGGATAAAACAGGTTTGGGTC-3′), as well as in the NADH dehydrogenase subunit 5 that was used as an internal control (Menzel et al. 2002). RT-PCR included an initial denaturation for 5 min at 95 °C and 34 cycles of 10 s at 95 °C, 20 s at 54 °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). Amplicons of the expected size (215 bp) were obtained from the eight pome fruit samples, confirming the DAS-ELISA results. Next, PCR products from each host (apple isolate Guba 230 and pear isolate Baku 66) were purified with ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA) and Sanger sequenced at the Cornell Biotechnology Resource Center (Ithaca, NY). Sequences of the Azerbaijani ASPV isolates were deposited in GenBank as accession numbers PZ878832 (isolate Guba 230) and PZ878833 (isolate Baku 66). Sequence analyses with the DNASTAR Lasergene software package (v17.3.0.57) showed high sequence identity between the two Azerbaijani ASPV isolates at the nucleotide (91.6-92.5%), and amino acid (95.7-100%) levels. A high nucleotide identity (92.5%) was also recorded between ASPV Azerbaijani isolates and isolates from other geographic origins such as South Korea isolate RD-2018-2 from Asian pear (LC455710), but a lower nucleotide identity was obtained with Iranian isolate T6Ma (KM243902) (80.5%) and Turkish E398 isolate (OP476709) (85.5%) from apple. To the best of our knowledge, this is the first report of ASPV in apple and pear trees in Azerbaijan. This result expands our knowledge on the distribution of ASPV. The occurrence of ASPV alongside apple chlorotic leaf spot virus (ACLSV, Mustafayev et al. 2025a), apple stem grooving virus (ASGV, Mustafayev et al. 2025b), prunus necrotic ringspot virus (Mustafayev et al. 2026a), prune dwarf virus (Mustafayev et al. 2026b) emphasizes the need for a pome and stone fruit certification program in Azerbaijan. This is more so because the pear and apple trees infected with ASPV were co-infected with ASGV and/or ACLSV, as shown by RT-PCR.