Molecular detection of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in Palestine

Abstract

Citrus hosts various phytopathogens that have impacted productivity, including viroids. Missing data on the status of viroids in citrus in Palestine were not reported. This study was aimed to detect any of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in the Palestinian National Agricultural Research Center (NARC) germplasm collection Field inspections found symptoms such as leaf epinasty; vein discoloration, and bark cracking on various citrus varieties. RT-PCR revealed a significant prevalence of CVd-IV; CEVd and CVd-III (47%, 31%, and 22%; respectively). CVd-III variants with 91.3% nucleic acid sequence homology have been reported. The sequence of each viroid were deposited in GenBank as (OP925746 for CEVd, OP902248 and OP902249 for CVd-III-PS-1 and -PS-2 isolates, and OP902247 for CVd-IV). This was the first to report three of citrus viroids in Palestine, appealing to apply of phytosanitary measures to disseminate healthy propagating materials free from viroids.

Figure 1

Inspected citrus trees were exhibited symptoms as leaf epinasty on Pomelo (A); cankers of bark on Clementine (B); and leaf discoloration on Lemon (C), of putatively viroid’s pathogeneses.

Figure 2

Specific sets of primers were able to amplify the expected portion of the citrus viroid species: CEVd (a); CVd-III (b); and CVd-IV (c) from infected citrus plant samples by single RT-PCR, as well as multiplex one. Plant rRNA primers (r) were applied to amplify portion of 18s rRNA as an internal positive control. 50 bp DNA Ladder RTU (Bio-Helix) was used as a molecular marker.

Figure 3

Prevalence of viroids in the citrus as detected by RT-PCR.

Figure 4

Phylogenetic analysis of (a) Citrus viroid IV (CVd-IV) [Accession No.: OP902247]; (b) Citrus viroid III (CVd-III) [Accession No.: OP902248 and OP902249] and (c) Citrus exocortis viroid (CEVd) [Accession No.: OP925746] sequences against corresponding viroids accessions available in GenBank. The phylogenetic tree was generated by MEGA-11 program with evolutionary history inferred by the Neighbor-Joining in the bootstrap clustering test of 1,000 replicates. The percentage of replicated trees less than 50% was collapsed.