Iresine viroid 1 (IrVd-1) (Spieker 1996) is a member of the genus Pospiviroid. Occurrence of IrVd-1 has so far been reported in Canada, Germany, India, the Netherlands, and Slovenia. We have previously developed a polyprobe (POSPIprobe) that in hybridization assays efficiently detects eight pospiviroids and that was predicted to detect also IrVd-1 (Torchetti et al. 2012), although in the absence of an IrVd-1 isolate in our collection, we could not put to test this prediction. In a survey carried out in the spring of 2014 in the frame of the project URCOFI (Campania region, Southern Italy) a total of 60 ornamental species (Euphorbia spp., Begonia spp., Baucarmea recurvata, Portulaca spp., Celosia cristata, and C. plumosa) were tested by tissue printing hybridization (TPH) with the POSPIprobe as reported previously (Lolic et al. 2007). Most samples tested negative, except nine asymptomatic plants of the species Portulaca (3 out of 4 tested plants), C. cristata (1 out of 5 tested plants), and C. plumosa (5 out of 11 tested plants). Northern-blot hybridization assays, performed according to Hajizadeh et al. 2012 using the POSPIprobe, showed the presence, in the TPH-positive plants, of a circular RNA with a molecular size of about 370 nt and its corresponding linear form, confirming that those plants were infected by a pospiviroid. When the same RNA preparations were examined by RT-PCR using generic primers designed to detect most pospiviroids (Bostan et al. 2004), an amplicon of about 220 bp was generated, the sequence of which was highly similar to that of IrVd-1 (NC003613). Based on this information, we corroborated the presence of IrVd-1 in Portulaca spp, C. plumosa, and C. cristata by designing two sets of adjacent and complementary primers (Ir1F: 5?-GGAGCTCGTCTCCTTCCTTTC-3? and Ir2R: 5?-TCCTGTTTCTTCCGCCGCG-3?) and (Ir5R: 5?-CCAGGTTTCCCCGGGGATC-3? and Ir6F: 5?-GGAGCGAACTCGGCAAGGAGG-3?) specific for this viroid that were used, separately, for amplifying, cloning, and sequencing full-length cDNAs from one representative plant of the three different host species. Collectively, a total of 22 full-length cDNA clones were sequenced, which showed sequences almost identical to each other (97 to 100%) and to the reference IrVd-1 variant (96 to 97%). A total of five IrVd-1 new variants were deposited in GenBank (KR020037 to KR020041). No significant clustering according to the geographic origin or the host species was observed when a phylogenetic analysis was performed using all IrVd-1 sequence variants reported in this and previous studies. To our knowledge, this is the first report of IrVd-1 in Italy and of C. cristata as a natural host of this viroid. Additionally, this study confirms the ability of the POSPIprobe to detect IrVd-1, expanding to nine pospiviroids the range of this polyprobe, and supports the usefulness of TPH as a preliminary approach for viroid detection.
First report of iresine viroid 1 in ornamental pants in Italy and of Celosia cristata as a novel natural host
Torchetti EM;Di Serio F;
2015
Abstract
Iresine viroid 1 (IrVd-1) (Spieker 1996) is a member of the genus Pospiviroid. Occurrence of IrVd-1 has so far been reported in Canada, Germany, India, the Netherlands, and Slovenia. We have previously developed a polyprobe (POSPIprobe) that in hybridization assays efficiently detects eight pospiviroids and that was predicted to detect also IrVd-1 (Torchetti et al. 2012), although in the absence of an IrVd-1 isolate in our collection, we could not put to test this prediction. In a survey carried out in the spring of 2014 in the frame of the project URCOFI (Campania region, Southern Italy) a total of 60 ornamental species (Euphorbia spp., Begonia spp., Baucarmea recurvata, Portulaca spp., Celosia cristata, and C. plumosa) were tested by tissue printing hybridization (TPH) with the POSPIprobe as reported previously (Lolic et al. 2007). Most samples tested negative, except nine asymptomatic plants of the species Portulaca (3 out of 4 tested plants), C. cristata (1 out of 5 tested plants), and C. plumosa (5 out of 11 tested plants). Northern-blot hybridization assays, performed according to Hajizadeh et al. 2012 using the POSPIprobe, showed the presence, in the TPH-positive plants, of a circular RNA with a molecular size of about 370 nt and its corresponding linear form, confirming that those plants were infected by a pospiviroid. When the same RNA preparations were examined by RT-PCR using generic primers designed to detect most pospiviroids (Bostan et al. 2004), an amplicon of about 220 bp was generated, the sequence of which was highly similar to that of IrVd-1 (NC003613). Based on this information, we corroborated the presence of IrVd-1 in Portulaca spp, C. plumosa, and C. cristata by designing two sets of adjacent and complementary primers (Ir1F: 5?-GGAGCTCGTCTCCTTCCTTTC-3? and Ir2R: 5?-TCCTGTTTCTTCCGCCGCG-3?) and (Ir5R: 5?-CCAGGTTTCCCCGGGGATC-3? and Ir6F: 5?-GGAGCGAACTCGGCAAGGAGG-3?) specific for this viroid that were used, separately, for amplifying, cloning, and sequencing full-length cDNAs from one representative plant of the three different host species. Collectively, a total of 22 full-length cDNA clones were sequenced, which showed sequences almost identical to each other (97 to 100%) and to the reference IrVd-1 variant (96 to 97%). A total of five IrVd-1 new variants were deposited in GenBank (KR020037 to KR020041). No significant clustering according to the geographic origin or the host species was observed when a phylogenetic analysis was performed using all IrVd-1 sequence variants reported in this and previous studies. To our knowledge, this is the first report of IrVd-1 in Italy and of C. cristata as a natural host of this viroid. Additionally, this study confirms the ability of the POSPIprobe to detect IrVd-1, expanding to nine pospiviroids the range of this polyprobe, and supports the usefulness of TPH as a preliminary approach for viroid detection.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
