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We investigated the presence of Hop stunt viroid (HSVd) in apple samples, showing dappling fruit symptoms, in the Maragheh area (Northwest Iran) by means of RT-PCR. The viroid was detected only in leaves collected from symptomatic trees and a 298bp amplicon (IR-Gala) was directly sequenced in both directions. Multiple sequence alignment and Blast analyzes revealed that IR-Gala isolate shares the highest identity with grapevine isolates from Brazil and China. Amongst Iranian isolates of HSVd available in the GenBank, this isolate had the highest identity with grapevine isolate of HSVd from Maragheh region. In Phylogenetic analysis by MrBayes, IR-Gala was clustered with grapevine isolates from Brazil, China and Iran and may suggest that HSVd-apple isolate could be originated from grapevine.

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Australian grapevine viroid (AGVd), an apscaviroid of the family Pospiviroidae, was recently identified in vineyards of southern Iran. It had a relatively wide host range and caused stunting, leaf deformation, mottling and vein clearing in experimental hosts upon mechanical inoculation of nucleic acid extracts or agroinfiltration of the viroid infectious cloned DNA. Predicted secondary structure of the AGVD-Ir showed a difference from the predicted structure of the type isolate in the viroid pathogenicity domain. Mutational analyses showed sequence changes introduced into that domain of the AGVD-Ir clone decreased the viroid’s replication efficiency in planta but did not show any effects on its movement.

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Hop stunt viroid (HSVd) isolates have been reported as the causal agent of citrus cachexia in Mazandaran Province and recently shown to be associated with yellow corky vein disease of sweet orange and split bark disorder of sweet lime in the Fars Province, Iran. In the present work isolation and partial characterization of viroids from citrus trees affected by gummy stem blight is reported from Kohgiluyeh–Boyerahmad (KB) Province of Iran. Fifteen samples of citrus trees from Dehdasht area (KB Province) showing bark necrosis, gum exudation and die-back as well as seven citrus symptompless trees from the same area were tested for the prevalence of viroids, through Reverse Transcription Polymerase Chain Reaction (RT-PCR) followed by sequencing of PCR products. They were also tested for Citrus tristeza virus through Double-Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA). Two variants of HSVd which differed from GenBank isolates in nucleotide sequence and two variants of Citrus Bent Leaf Viroid (CBLVd) were identified in any of the symptomatic samples. Moreover, a Citrus Exocortis Viroid (CEVd) was found only in symptomatic sweet lime. An HSVd isolate from KB (HSVd-bn₁) was selected and used for comparison with a number of HSVd variants from Iran (Fars and Mazandaran Provinces) and the related accessions from GenBank. On the basis of nucleotide sequence and secondary structure analysis, HSVd-bn1 and HSVd-bn2 belong to non-cachexia variants of HSVd and have about 95% similarity to Citrus gummy bark viroid, a sub-species of HSVd. CTV was not detected in the diseased plants. It is yet to be determined whether bark necrosis of sweet lime and of sweet orange plants is caused solely by the associated viroid(s) or other factors are involved as well.

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Citrus plants are hosts of several viroid species, among which, pathogenic variants of Hop Stunt Viroid (HSVd) induce citrus cachexia disease. Stunting, chlorosis, gumming of the bark, stem pitting and decline are symptoms of cachexia in mandarins and their hybrids as susceptible hosts. Based on the pathogenic properties on citrus, HSVd variants are divided in two distinct groups: those that are symptomless on sensitive citrus host species and those that induce cachexia disease. In this study, two cachexia isolates were selected and biological indexing was performed in a controlled temperature greenhouse (40ºC day and 28ºC night) using Etrog citron (Citrus medica) grafted on Rough lemon (C. jhambiri), as a common indicator for citrus viroids. The plants were inoculated with the inocula from a severe symptomatic tree of a newly declining orchard of Jiroft, Kerman province and a mild symptomatic tree from Mazandaran province. Presence of HSVd was confirmed with sPAGE, Hybridization by DIG-labeled probes and RT-PCR using specific primers of HSVd. Primary and secondary structures of the isolates were studied. The consensus sequence of RT-PCR amplicons of the severe isolate (JX430796) presented 97% identity with the reference sequence of a IIb variant of HSVd (AF213501) and an Iranian isolate of the viroid (GQ923783) deposited in the gene bank. The mild isolate (JX430798) presented 100% homology with the HSVd-IIc variant previously reported from Iran (GQ923784). Both isolates were shown to be cachexia inducing according to their sizes, sequences and lack of “non-cachexia expression motif” structures.

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Citrus Viroid V (CVdV) is a member of the genus Apscaviroid, in the Pospiviroidae family. It is restricted to citrus species naturally. The herbaceous host range of CVdV was determined using the viroid infectious clone. Several herbaceous plants from the Cucurbitaceae, Solanaceae, Fabaceae, and Asteraceae families were found to be susceptible to CVdV. Also, CVdV could be transmitted to these hosts through rubbing of monomeric DNA plasmids and through mechanical inoculation of infected sap. The accumulation of CVdV in the tomato was monitored up to 28 days after inoculation and a further 56-fold increase of viroid titer was observed. Analysis of sequences of the viroid progenies from herbaceous plants revealed several nucleotide substitutions, which mostly concentrated in the pathogenicity domain on the secondary structure of the viroids.