Abstract
Apple chlorotic leaf spot virus (ACLSV) is a globally distributed pathogen affecting apple trees and other members of the Rosaceae family, with implications for fruit yield and quality. This study represents the comprehensive investigation of ACLSV presence in both cultivated Malus domestica and wild Malus sieversii apple populations across southeastern Kazakhstan for the first time. A total of 419 leaf and branch samples from five distinct locations were screened using newly developed primers for conventional and real-time RT-PCR, with confirmation via digital PCR and a novel CRISPR/Cas12a-based detection assay. ACLSV was detected in 53.2% of the samples, including populations of M. sieversii not affected by human influence. Selected ACLSV-positive samples were subjected to nanopore sequencing, revealing multiple viral haplotypes (quasispecies) within individual hosts. Phylogenetic analysis indicated complex genetic relationships between local and homologous sequences from GenBank, suggesting ongoing bidirectional transmission. Recombination analysis identified multiple events within the coat and movement protein regions. The CRISPR/Cas12a assay showed high sensitivity and specificity, offering a potential alternative to PCR for field diagnostics. These findings highlight the widespread presence and genetic diversity of ACLSV in Kazakhstan and emphasize the importance of molecular surveillance in both cultivated and wild apple populations.