Citrus plants are infected by at least seven viroid species belonging to thePospiviroidae family (Murcia et al., 2015). In Chania prefecture (Crete island), Citrusspp. are considered as important cultivated species and especially the Persian (orTahiti) lime (Citrus latifolia) has become a new dynamic and expanding crop. Viroidshave been reported in other citrus species in Greece (Boubourakas et al., 2010; Wanget al, 2013), but no research has been conducted in lime. In the spring of 2018, in asmall lime orchard of forty 5-years old trees (grafted on C. aurantium), yellow fruitsectoring and shoot bark cracking symptoms were observed in six trees. A total ofeight samples, six symptomatic and two asymptomatic trees, were collected. All ofthem were tested for the presence of the most prevalent citrus viroid, citrus exocortisviroid (CEVd) by reverse transcription-polymerase reaction (RT-PCR), amplifyingthe entire viroid genome as previously described (Bernad and Duran-Villa, 2006). Theresults revealed the presence of CEVd in all the symptomatic plants, whereas theviroid was not detected in the asymptomatic trees. In an effort to study the virome oflime species, one symptomatic sample (Cl-0) was subjected to high-throughputsequencing (HTS) of total RNA extracted from phloem tissue, on an IlluminaNextSeq 500 platform, generating ~12.5 million of 150 bp pair-end reads. The denovo assembly of these reads and the BLASTN analysis of the produced contigsresulted in the confirmation of CEVd presence in addition to the identification of hopstunt viroid (HSVd), citrus bark cracking viroid (CBCVd) and citrus dwarfing viroid(CDVd). A recently discovered virus was also detected, which will be describedelsewhere. The complete viroid genomes of 370, 302, 286 and 291 nucleotides inlength for CEVd, HSVd, CBCVd and CDVd, respectively, were reconstructed and thegenomes shared 99% (CEVd, CDVd) and 100% (HSVd, CBCVd) nucleotide identitywith other available sequences of isolates from different countries. To confirm theresults obtained from the HTS experiment, the Cl-0 sample in addition with allsymptomatic (six in total) and two asymptomatic trees were further tested for thepresence of HSVd, C?CVd and CDVd using specific primers to amplify the fullviroid genome, as previously reported (Bernad and Duran-Villa, 2006). The RT-PCRresults revealed the presence of HSVd also in all six symptomatic trees, whereasCDVd and CBCVd were detected in five and four symptomatic samples, respectively.The mixed infections in the symptomatic trees were with three or four viroids. Noviroid was detected in the asymptomatic trees. Direct Sanger sequencing of the PCRamplicons confirmed the presence of the four viroids in the HTS analyzed Cl-0sample. The retrieved nucleotide sequences were identical to those recovered from theHTS, and were deposited in GenBank with accession numbers MN136643,MN136644, MN136645 and MN136646. To our knowledge, this is the first report ofcitrus viroids infecting lime in Greece associated with bark cracking symptoms andongoing studies with larger surveys and HTS are in progress to elucidate a possiblelink, if any, between symptoms and presence of a specific viroid.
First Report of Citrus Viroids Infecting Persian (Tahiti) Lime in Greece
Chiumenti M;
2020
Abstract
Citrus plants are infected by at least seven viroid species belonging to thePospiviroidae family (Murcia et al., 2015). In Chania prefecture (Crete island), Citrusspp. are considered as important cultivated species and especially the Persian (orTahiti) lime (Citrus latifolia) has become a new dynamic and expanding crop. Viroidshave been reported in other citrus species in Greece (Boubourakas et al., 2010; Wanget al, 2013), but no research has been conducted in lime. In the spring of 2018, in asmall lime orchard of forty 5-years old trees (grafted on C. aurantium), yellow fruitsectoring and shoot bark cracking symptoms were observed in six trees. A total ofeight samples, six symptomatic and two asymptomatic trees, were collected. All ofthem were tested for the presence of the most prevalent citrus viroid, citrus exocortisviroid (CEVd) by reverse transcription-polymerase reaction (RT-PCR), amplifyingthe entire viroid genome as previously described (Bernad and Duran-Villa, 2006). Theresults revealed the presence of CEVd in all the symptomatic plants, whereas theviroid was not detected in the asymptomatic trees. In an effort to study the virome oflime species, one symptomatic sample (Cl-0) was subjected to high-throughputsequencing (HTS) of total RNA extracted from phloem tissue, on an IlluminaNextSeq 500 platform, generating ~12.5 million of 150 bp pair-end reads. The denovo assembly of these reads and the BLASTN analysis of the produced contigsresulted in the confirmation of CEVd presence in addition to the identification of hopstunt viroid (HSVd), citrus bark cracking viroid (CBCVd) and citrus dwarfing viroid(CDVd). A recently discovered virus was also detected, which will be describedelsewhere. The complete viroid genomes of 370, 302, 286 and 291 nucleotides inlength for CEVd, HSVd, CBCVd and CDVd, respectively, were reconstructed and thegenomes shared 99% (CEVd, CDVd) and 100% (HSVd, CBCVd) nucleotide identitywith other available sequences of isolates from different countries. To confirm theresults obtained from the HTS experiment, the Cl-0 sample in addition with allsymptomatic (six in total) and two asymptomatic trees were further tested for thepresence of HSVd, C?CVd and CDVd using specific primers to amplify the fullviroid genome, as previously reported (Bernad and Duran-Villa, 2006). The RT-PCRresults revealed the presence of HSVd also in all six symptomatic trees, whereasCDVd and CBCVd were detected in five and four symptomatic samples, respectively.The mixed infections in the symptomatic trees were with three or four viroids. Noviroid was detected in the asymptomatic trees. Direct Sanger sequencing of the PCRamplicons confirmed the presence of the four viroids in the HTS analyzed Cl-0sample. The retrieved nucleotide sequences were identical to those recovered from theHTS, and were deposited in GenBank with accession numbers MN136643,MN136644, MN136645 and MN136646. To our knowledge, this is the first report ofcitrus viroids infecting lime in Greece associated with bark cracking symptoms andongoing studies with larger surveys and HTS are in progress to elucidate a possiblelink, if any, between symptoms and presence of a specific viroid.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
