First Report of Apple Chlorotic Leaf Spot Virus in Pome and Stone Fruit Trees in Azerbaijan.

Azerbaijan is major producer of fruit crops, such as pome and stone fruits, in the Caspian Sea and Caucasus Mountains areas (FAO Stat, 2022). No information is available on the occurrence of apple chlorotic leaf spot virus (ACLSV, genus Trichovirus, family Betaflexiviridae) in the country. Therefore, the main fruit tree production areas in Azerbaijan were surveyed for ACLSV during the 2017-2019 growing seasons by DAS-ELISA using ACLSV reagents (Neogen - Scotland, UK) (Clark and Adams 1977). Of the 510 plant samples tested, 139 were positive for ACLSV. To confirm the presence of ACLSV, total RNA was isolated from leaf samples (0.5 g) exhibiting deformation, yellow veins, ringspots, and/or chlorosis symptoms 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 ACLSV: F (5'-CTACAATTTGAAGGAGGTGGTCAAC-3'), R (5'-GACGCCGGTTCATATTAGTTATGAC-3') and primers for the NADH dehydrogenase subunit 5 fragment as a housekeeping gene (Menzel et al. 2002). Reactions were performed at 5 min at 95 °C, 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). Results showed the occurrence of ACLSV in 14 apple samples (Malus domestica L.; n=38), four cherry plum sample (P. cerasifera L.; n=17), one almond samples (P. amygdalus L.; n=13), and one plum sample (P. domestica L.: n=71). All the pear (Pyrus communis L.; n=27), quince (Cydonia oblonga L.; n=2), sweet cherry (P. avium L.; n=27), sour cherry (P. cerasus L.; n=1), peach (P. persica L.; n=22), and apricot (P. armeniaca L.; n=8) samples tested negative for ACLSV in RT-PCR. Next, the 287-bp amplicons obtained by RT-PCR from two Azerbaijani ACLSV isolates from the Ganja and Kurdamir region, i.e., Ganja 43 from apple and Kurdamir 80 from plum, were cleaned with Exo SAP-IT and characterized by bidirectional Sanger sequencing at the Cornell Biotechnology Resource Center in Ithaca, NY. Sequence analyses were performed with the DNASTAR Lasergene software package (v17.3.0.57). Results revealed 87.2%-96.7% and 98.3%-100% nucleotide and amino acid identities, respectively, of ACLSV isolate Ganja 43 isolate (GenBank PP894314) with Afghan (KU749440) and Ukrainian (JQ866623) isolates, and 86.6%-95.2% and 97.9%-99.0% nucleotide and amino acid identities, respectively, of ACLSV isolate Kurdamir 80 (GenBank PP898052) with Afghan (KU749440) and Indian (FN666579) isolates. This is the first report of ACLSV in pome and stone fruit trees in Azerbaijan. Since the occurrence of mixed virus infections was not investigated in this study, the viral symptoms displayed by the infected trees cannot be ascribed to ACLSV. The occurrence of ACLSV suggests the need for certification programs in the country to reduce the presence of the virus in the propagation material of pome and stone fruit trees.