Leaf yellowing and reddening symptoms on Vitis vinifera vines were observed in different grapevine nurseries in the province of Kurdistan in the west of Iran during 2016-2017. Total DNA was extracted from 27 symptomatic and four symptomless plants and tested for the presence of phytoplasma DNA by nested polymerase chain reaction (PCR) using P1/P7 and R16F2n/R16R2 primer pairs. PCR amplicons of about 1.8 and 1.2 kb in size were obtained from all symptomatic V. vinifera but not from symptomless plants. Restriction fragment length polymorphism (RFLP) profiles of R16F2n/R2 amplicons using AluI, HhaI, HpaII, RsaI, TaqI, KpnI, HaeIII, and Sau3AI restriction enzymes were identical to each other and to those obtained from 16SrI phytoplasmas, indicating that the phytoplasmas associated with grapevine yellowing and reddening were members of the 16SrI group. A R16F2n/R16R2 16S rDNA sequence of Kurdistan V. vinifera yellowing and reddening strain (KVvY) was submitted to GenBank as accession number MH638316. The Blast analysis of KVvY showed 100% sequence identity with those of the ‘Candidatus Phytoplasma asteris’-related strains. Phylogenetic analysis confirmed that the phytoplasma associated with KVvY clustered within the 16SrI phytoplasma clade closer to the Onion yellows mild strain (OY-M), a 16SrI-B phytoplasma. Restriction analysis using iPhyClassifier confirmed that virtual RFLP patterns of the KVvY were identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession AP006628).

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.

During 2016-2017 surveys, carried out for phytoplasma diseases in ornamental plants in Chaharmahal and Bakhtiari provinces, Iran, found symptoms of virescence, phyllody, reduced size of leaves and flowers in columbine (Aquilegia vulgaris). Total DNAs extracted from symptomatic and symptomless plants were tested for the presence of phytoplasma using P1/P7 and R16F2n/R16R2 primers in direct and nested PCR producing amplicons of about 1.8 and 1.2 kb, respectively, from all symptomatic A. vulgaris plants, but not from symptomless ones. The consensus sequence of the detected phytoplasma named Aquilegia phyllody (APh) was 100% identical with strains clustering to phytoplasmas enclosed in the 16SrI group as also confirmed by phylogenetic analyses. Both real and virtual restriction fragment length polymorphism analysis of R16F2n/R16R2 amplicons showed profiles that were identical to each other and indicated the affiliation of the APh phytoplasma to the 16SrI-R subgroup. This is the first report of a 16SrI-R phytoplasma associated with this A. vulgaris phyllody disease.

Tamarisk witches’ broom, yellowing, and little leaf symptoms were observed during 2018–2023 surveys of rural deserts in central regions of Iran with the highest disease incidence up to 72% in Chah Afzal (Yazd province). A verification of the presence and identity of phytoplasmas associated with these symptoms was then performed. Tamarisk tree branch cuttings obtained from symptomatic plants sprouted up to 90.3% but with 15–25 days’ delay compared to the asymptomatic ones and showed internode shortening and witches’ broom, while the branch cuttings from asymptomatic plants had normal growth and sprouted up to 97.8%. Phytoplasma transmission by dodder bridges to periwinkle did not succeed, while nested polymerase chain reaction on the phytoplasma ribosomal gene followed by RFLP and phylogenetic analyses revealed the presence of ‘Candidatus Phytoplasma asteris’, ‘Ca. P. australasiae=australasiaticum’, and ‘Ca. P. trifolii’ (ribosomal subgroups 16SrI-B, 16SrII-D, and 16SrVI-A, respectively) in the samples from symptomatic plants only. Further amplifications were performed on selected phytoplasma-positive samples on tuf and secA genes, and the produced sequences indicated the presence of mixed phytoplasma infection in some of the samples. In particular, in the tuf gene, a mixed infection of ‘Ca. P. australasiae=australasiaticum’ and ‘Ca. P. trifolii’ was detected, while in the secA gene, the presence of ‘Ca. P. asteris’ or ‘Ca. P. tritici’ strains was identified. The first-time detection of diverse phytoplasma strains in symptomatic T. aphylla suggests that this species represent a relevant source of infection for the agricultural crops and for landscape plants especially when temperature allows insect vector transmission, and therefore, it represents a risk in every environment especially in the frame of climatic changes.

Background: Circularization of RNA mediated by association of translation factors and RNA elements in 5′ and 3′ UTRs is a common feature for translation control in eukaryotes. There is no information about translation in plant rhabdoviruses and little information is known in animal rhabdoviruses. Methods: The role of 5′ and 3′ UTRs in two genes of EMDV in translation were studied using luciferase constructs and RNA structures of these sequences were analyzed by SHAPE and Inline probing. Results: We have found that efficient translation of N and X mRNAs of nucleorhabdovirus Eggplant mottled dwarf virus (EMDV) requires elements present in both 5′ and 3′ UTRs. Luciferase reporter constructs containing precise 5′ and 3′ UTRs of the N and X genes had substantially higher translational activity compared with constructs containing only the 5′ or 3′ UTR. The 3′UTR of carmovirus Turnip crinkle virus, which contains a well-characterized cap-independent translation enhancer, was unable to complement the lack of EMDV 3′ UTR. Addition of cap analog to luciferase constructs containing the UTRs of the N gene did not restore translation, and translation of the reporter construct in the absence of the 5′ cap was higher than the capped construct. No RNA-RNA interactions between 5′ and 3′ UTRs were detected by EMSA or in-line cleavage structural assays. Deletion of 11 nucleotides from the 3′ terminus negated the synergistic activity of the 3′UTR. Conclusions: The results with RNA-RNA interaction suggesting that translational synergy between the UTRs may utilize alternative means. Mutation analysis in 3′UTR suggesting that the polyadenylation signal sequence contained in this location may play a critical role in translation.