Prevalence and molecular variability of Apple chlorotic leaf spot virus capsid protein genes in Lithuania

Apple chlorotic leaf spot virus (ACLSV) is a most common pathogen of apples in the world. The occurrence, genetic diversity, recombination patterns and mutation of ACLSV CP in apple trees 80-120 years old from old homesteads and abandoned orchards in Lithuania were investigated. Using RT-PCR, 73.8% of samples tested positive for ACLSV. Specific PCR products to virus CP gene were sequenced and analysed from 39 RNA samples of apple trees collected in different geographic locations. Heterogenic infection of ACLSV in the same apple tree was detected in 11 samples. Fifty Lithuanian sequences of ACLSV with diversity in CP gene were submitted in NCBI and compared to 28 analogous sequences by other authors. The identity of virus CP gene among Lithuanian isolates ranged 81.6-99.7% at the nucleic and 88.6-100% at the amino acids level. ACLSV isolates significantly distinguished into two clusters and seven subclusters on phylogenetic tree. Isolates related to P205, A4, B6 and GC10a were identified in Lithuania, and relationship with Mo-5 and RC was not found. Two recombinant Lithuanian isolates of ACLSV distinguished to separate subcluster R-LT. Lithuanian isolates were characterized by combination of amino acids in conservative sites. The unique single or multiple mutations in CP gene among Lithuanian isolates were observed, and substitution in conservative sites 59, 75 and 79 was also identified. The high prevalence and diversity of latent infection in old apple trees was frequently determined due to the genomic mutation and rarely due to the recombination.