Detection of citrus leaf blotch virus in sweet orange in Croatia, using metagenomics and extraction-free RT-PCR

In late 2022, uncharacteristically small fruits of Washington Navel sweet orange (Citrus sinensis L. ‘Washington Navel’) with fruit creasing symptoms were sampled in a small orchard on the coast of Croatia. The virome in the fruits was investigated using metagenomic analysis, which showed presence of citrus dwarfing viroid (CDVd) and citrus leaf blotch virus (CLBV). The record of CLBV is the first for Croatia. Nucleotide sequence analysis of the RdRp region indicated that the Croatian CLBV isolate shared closest sequence similarity to two isolates from kumquat Nagami from Sicily. Full-genome phylogeny of CLBV accessions indicate that isolates from citrus cluster together and are mostly distinct from isolates from non-citrus hosts. The use of Fast Technology for Analysis (FTA) cards was evaluated, as an alternate for CLBV sampling, and as a transport-safe method for obtaining viral RNA templates for RT-PCR. FTA cards in combination with an extraction-free protocol were shown to be usable when columella tissue was used to imprint, but leaf prints were unreliable.