First Report of Citrus Viroids Infecting Persian (Tahiti) Lime in Greece

Citrus plants are infected by at least seven viroid species belonging to the Pospiviroidae family (Murcia et al., 2015). In Chania prefecture (Crete island), Citrus spp. are considered as important cultivated species and especially the Persian (or Tahiti) lime (Citrus latifolia) has become a new dynamic and expanding crop. Viroids have been reported in other citrus species in Greece (Boubourakas et al., 2010; Wang et al, 2013), but no research has been conducted in lime. In the spring of 2018, in a small lime orchard of forty 5-years old trees (grafted on C. aurantium), yellow fruit sectoring and shoot bark cracking symptoms were observed in six trees. A total of eight samples, six symptomatic and two asymptomatic trees, were collected. All of them were tested for the presence of the most prevalent citrus viroid, citrus exocortis viroid (CEVd) by reverse transcription-polymerase reaction (RT-PCR), amplifying the entire viroid genome as previously described (Bernad and Duran-Villa, 2006). The results revealed the presence of CEVd in all the symptomatic plants, whereas the viroid was not detected in the asymptomatic trees. In an effort to study the virome of lime species, one symptomatic sample (Cl-0) was subjected to high-throughput sequencing (HTS) of total RNA extracted from phloem tissue, on an Illumina NextSeq 500 platform, generating ~12.5 million of 150 bp pair-end reads. The de novo assembly of these reads and the BLASTN analysis of the produced contigs resulted in the confirmation of CEVd presence in addition to the identification of hop stunt viroid (HSVd), citrus bark cracking viroid (CBCVd) and citrus dwarfing viroid (CDVd). A recently discovered virus was also detected, which will be described elsewhere. The complete viroid genomes of 370, 302, 286 and 291 nucleotides in length for CEVd, HSVd, CBCVd and CDVd, respectively, were reconstructed and the genomes shared 99% (CEVd, CDVd) and 100% (HSVd, CBCVd) nucleotide identity with other available sequences of isolates from different countries. To confirm the results obtained from the HTS experiment, the Cl-0 sample in addition with all symptomatic (six in total) and two asymptomatic trees were further tested for the presence of HSVd, C?CVd and CDVd using specific primers to amplify the full viroid genome, as previously reported (Bernad and Duran-Villa, 2006). The RT-PCR results revealed the presence of HSVd also in all six symptomatic trees, whereas CDVd and CBCVd were detected in five and four symptomatic samples, respectively. The mixed infections in the symptomatic trees were with three or four viroids. No viroid was detected in the asymptomatic trees. Direct Sanger sequencing of the PCR amplicons confirmed the presence of the four viroids in the HTS analyzed Cl-0 sample. The retrieved nucleotide sequences were identical to those recovered from the HTS, and were deposited in GenBank with accession numbers MN136643, MN136644, MN136645 and MN136646. To our knowledge, this is the first report of citrus viroids infecting lime in Greece associated with bark cracking symptoms and ongoing studies with larger surveys and HTS are in progress to elucidate a possible link, if any, between symptoms and presence of a specific viroid.