akbar dizadji

Potato virus Y (PVY) is one of the viruses limiting tobacco and potato production in the world. To investigate the biological and molecular characteristics of PVY isolates, during 2010-2011, a total of 1178 symptomatic leaf samples of tobacco, potato, pepper and grand cherry (Physalis divaricata) plants were collected from eight different cities including Karaj, Urmia, Sanandaj, Shiraz, Qazvin, Varamin and Hamedan of Iran. The samples were tested serologically by double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) and PVY was detected in tobacco (27.27 %), potato (20.4 %) and grand cherry (18.75 %) samples, with no infection in pepper samples. Four biologically purified PVY isolates (Po2, Po5, Tob2 and Ph1) revealed few differences in experimental host range studies among 12 test plant species. After amplification of the 3´ genomic end of four PVY isolates with the expected size of 1200 bp, phylogenetic analysis based on the nucleotide sequences of partial coat protein gene (encoding N- terminal region of coat protein) revealed that Tob2, Po5 and Ph1 isolates grouped into PVYN/NTN clade, while only isolate Po2 grouped into PVYO clade. The results of IC-RT-PCR using strain-specific primers and further pairwise nucleotide sequence comparisons and strain-specific motifs analysis in the predicted amino acid sequences of the N-terminal region of coat protein confirmed the results of phylogenetic analysis. This is the first report of Physalis divaricata infection with PVYN/NTN strain in the world.

Nucleic acid-based diagnostic approaches are significant methods in detection and identification of viruses due to their small genome structures and minimal nucleotide differences among virus isolates or strains. In this study, dead or diseased larvae of Helicoverpa sp. were collected from tomato fields in distinct geographic areas in Iran and baculovirus infection was assayed by occlusion body extraction using an optimized Sodium Dodecyl Sulphate method. Two sets of specific polymerase chain reaction (PCR) primer pairs (Pol-a and Pol-b) were designed based on the conserved polyhedrin gene region of 26 fully sequenced HearSNPV/HzNPV isolates. Accordingly, infection by HearSNPV was confirmed in 28 out of 34 tested larvae by PCR amplification of monomorphic fragments of about 370 and 790 nucleotides, respectively. Cloning and sequencing of fragments showed that they belong to the corresponding polyhedron gene from nucleopolyhedriviruses of H. arimegra and H. zea species with 99.6% sequence identity. The phylogeny trees developed for Iranian isolates based on their polyhedron gene sequences showed that they all belong to the Group II Alphabaculovirus and formed one clade with other HearSNPV isolates. This, further confirmed the efficiency of the developed method for baculovirus detection and characterization. To our knowledge, this is the first report of introduction and molecular characterization of some HearSNPV isolates, circulating in these pests in Iran, using a robust DNA-based approach. The excellent efficacy of this method in virus detection makes it a valuable tool for rapid screening of insect cadavers for HearNPV infection, phylogenic studies and virus monitoring in bioinsecticides application.

Potato virus Y, is the type member of the genus Potyvirus, family Potyviridae is of the most destructive viruses of tobacco worldwide. In order to detect PVY and its distribution in Golestan province, several surveys were conducted during July to October 2017 at 15 days intervals in tobacco fields of Gorgan, Aliabad and Minoodasht. 50 plant was selected for each farm and various disease indicators and the virus vector (MP and MPN( were evaluated. Double antibody sandwich-ELISA (DAS-ELISA) using PVY-specific polyclonal antibodies was used to estimate changes in the relative concentrations of PVY. The rate of PVY infection was different among tobacco cultivars and the maximum and minimum infection rates were belonged to PVH03 (33.6%) and Barley (6.2%) cultivars respectively. Based on the statistical analysis of data, there was a significant difference for disease indicators and the virus vector on different tobacco cultivars, as Basma and Barley were less susceptible to PVY than Flue-cured cultivars (PVH03, NC100 and K326). Also, the K326 cultivar has the highest Myzus persicae ssp. nicotiana and Myzus persicae populations. There was a direct correlation between DI and OD and relative humidity and rainfall changes so that DI with rainfall changes had the highest Correlation (r = 0.98 and P = 0.004).

Faba bean necrotic yellows virus (FBNYV) causes severe yield losses and crop failure in legumes in the north African, south Asian and European countries. Following nucleotide sequencing of DNA-M segment of 17 host and geographical FBNYV isolates in Iran, different molecular analysis were performed based on all available DNA-M sequences of FBNYV. Based on constructed phylogenetic tree using nucleotide sequence of DNA-M component, FBNYV isolates were grouped into three clades, including isolates from Iran and Azerbaijan as a distinct clade. Genetic distance studies of FBNYV host subpopulations revealed some evidences of host adaptation in FBNYV natural selection. High gene flow was found among Iran – Azerbaijan and Spain – Africa subpopulations, indicating the relationship of their origins. Maximum genetic diversity of DNA-M was estimated in the central subpopulation of Iran. The dN/dS (Ѡ) value less than 1 represented the negative selection pressure on the movement protein of the virus. Seven out of nine recombination events were detected in DNA-M of Iranian FBNYV isolates. This is the first report of the FBNYV infection on pea from Iran. In the current study, the complete DNA-M nucleotide sequences of French bean and pea isolates of FBNYV were determined for the first time in the world.

Interaction among two isolates of Cucumber mosaic virus </em>(CMV), Zin1 and Zin2 belong to CMV subgroup I and II was investigated on Nicotiana rustica </em>plants in current study. Following singly and doubly mechanical inoculation of pure isolates on N. rustica </em>plants, infection rate (frequency of detection) and relative concentration of each CMV isolate were calculated in six different time trials (days post inoculation, dpi) by quantitative ELISA. Statistical analysis of repeated measures data showed a meaningful effect (p<0.05) of treatment and time variants on frequency of detection and relative concentration of each isolates. D/S ratio for each CMV isolate was less than 1 in doubly inoculation treatment. The results showed that the relative concentration of each isolate in singly inoculated plants was significantly (p<0.01) more than doubly inoculated ones at 5, 10 and 18 dpi. The increase in virus titre at 18 dpi was confirmed by western blot analysis. These results demonestrated a cross-protection interaction among CMV subgroups. Variance analysis of data showed that unlike area under the disease progress curve (AUDPC) and concentration of chlorophyll, disease severity index (DSI), height and fresh weight of plants showed a significant difference among different tratments. In aphid transmission assays by Aphis gossypii </em>fed on singly and doubly infected source plants, no significant difference was observed among transmission rate (T) and single aphid transmission efficiency (P*</sup>) of each CMV isolates.

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease, the economically important viroid disease of citrus. In this research, sequence polymorphism and population structure of CEVd were investigated in CEVd tolerant and sensitive hosts (sour orange (Citrus aurantium L.) and citrange (Poncirus trifoliata (L.) Raf. × C. sinensis (L.), respectively) seedlings. Full-length in vitro transcript of single sequence CEVd-S1 isolate was used for mechanical inoculation. The genetic diversity of CEVd populations was estimated in two citrus hosts by single-strand conformation polymorphism (SSCP) and sequencing. The analysis of cloned DNAs recovered from infected hosts by this isolate demonstrated that host species was effective on the variability within a single CEVd isolate. The amount and composition of the genetic diversity were different among the two hosts, and was higher in the tolerant host compared with the sensitive one. Furthermore, the analysis of thermodynamic secondary structures illustrated that nucleotide changes identified in this study did not induce major modifications in the viroid rod-like secondary structure.

Cucumber mosaic virus (CMV; genus Cucumovirus, family: Bromoviridae) has the widest host range of any known plant viruses. Seven virus isolates, originated from different ornamental plant species and greenhouses, were biologically purified, mechanically inoculated onto test plants and their serological differences were assayed based on reactivity with 11 CMV-specific monoclonal antibodies. Following total RNA extraction, coat protein (CP) coding region of CMV isolates was amplified. Based on biological, serological and phylogenetic analysis, only one isolate belonged to CMV subgroup II and other six isolates were equally distributed among the two IA and IB subgroups. Aphid transmission assay showed that no significant difference was observed between transmission efficiency of CMV subgroups IA, IB and II members by Aphis gossypii. The genetic variation and evolution of CMV in Iran was studied by sequence analysis of the CP gene and comparison with equivalent sequences of isolates from other continents that exhibited low genetic diversity and close evolutionary relationships among isolates in subpopulations. Analysis of various population genetics parameters and distribution of synonymous and nonsynonymous mutations revealed that most of the amino acid sites were under negative selection and only one site was under positive selection.

Tomato yellow leaf curl virus (TYLCV) is a supreme pathogen in tropical and subtropical areas. During 2014-2015, a total of 393 tomato samples showing Tomato yellow leaf curl disease (TYLCD) symptoms were collected from six different provinces of Iraq. In serological assays, 55 out of 393 samples (14%) reacted positively with TYLCV-specific antibodies .The presence of TYLCV was verified in 21 (out of 55 samples showing tomato yellow leaf curl disease) samples by PCR. Coat protein gene (V1) of 16 TYLCV isolates was amplified using specific primers and cloned. Nucleotide sequence of Iraqi TYLCV isolates V1 gene shared the highest nucleotide sequence identity of 94.1-99.6% in CP gene with Kuwait and Iraq isolates. In phylogenetic analysis based on V1 nucleotide sequences, Iraqi isolates were separated into two main groups and three subgroups, without correlation with geographical origin. Genetic diversity parameters based on V1 nucleotide sequences indicated a high genetic variability in Iraqi population of TYLCV. The proportion of non-synonymous to synonymous nucleotide diversity was <1.0, indicating that this gene is under negative selection. A low gene flow was observed between southern and centric Iraq subpopulations, demonstrating genetic differentiation among Iraqi subpopulations. This is the first study dealt with distribution and genetic variation of TYLCV in Iraq. Full length viral genome sequencing of TYLCV isolates of Iraq is necessary to defferentiate virus strain(s) in Iraq.

Following the random sampling of saffron (Crocus sativus L.)leaves and corms from six provinces of Iran, during growing seasons of 2011-2015, a total of 890 leaf tissue samples were investigated by enzyme-linked immunosorbent assay (ELISA) tests. The results revealed that 641 samples showed positive reaction only with Potyvirus genus specific and exactly with Bean common mosaic virus (BCMV) specific antibodies. The molecular mass of several isolates of the virus coat protein (CP) was estimated as 35 kDa by western blotting. Following total RNA extraction from virus infected samples, three discontinuous fragments (corresponding to genomic 3', partial CI and HC-Pro regions) were amplified. The CP region of the virus infecting saffron has the highest nucleotide identity with Ceratobium mosaic virus (CerMV) and Telosma mosaic virus (TelMV) (74%) and the highest deduced amino acid identity with Bean common mosaic necrosis virus (BCMNV) (78.8%). Despite the high serological relationship with BCMV and a close phylogenetic relationship with BCMV subgroup based on CP region, the identity values are less than described species demarcation criteria for Potyviridae family. Overall, it seems that the saffron infecting Potyvirus is a new species of Potyvirus genus but the definitive statement requires sequencing the entire genome of the virus.

Bean yellow mosaic virus (BYMV, Potyvirus, Potyviridae) causes serious disease in Gladiolus spp. In this work, the possibility of obtaining BYMV free plant material from virus infected gladiolus corms was studied. Thermotherapy, meristem-tip culture and combination of both techniques on infected corms/meristem-tip explants (0.5–1 mm in length) resulted in BYMV elimination up to 15.38, 78.04 and 86.66%, respectively, as determined by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (IC-RT-PCR). Individual virus-free shoots readily rooted in vitro and were transferred to corm formation medium. The results showed that thermotherapy promotes the survival rate of explants during meristem-tip culture steps (except regeneration step) and also plantlet acclimatization. Statistical analysis showed that the BYMV elimination in gladiolus corms was significantly (P ≤ 0.01) affected by thermotherapy treatment of infected corms. Thermotherapy combined with meristem culture can greatly improve BYMV elimination efficiency from infected gladiolus corms, resulting in the production of BYMV free gladiolus plants.

Potato virus Y (PVY) is one of the most important plant viruses affecting tobacco fields in Golestan، Mazandaran and Guilan provinces (Iran). Given the lack of insecticide impact in reducing infection to PVY، breakdown of the natural resistance resources by certain strains and the problems with traditional breeding methods، the use of alternative methods to generate PVY resistant tobacco by pathogen-derived resistance could be considered as a method for reducing the damages. The objective of this study was to produce transgenic tobacco plants with resistance against a diverse spectrum of PVY isolates from different strains. A 472 bp fragment of the genome of an Iranian PVY isolate including the partial nucleotide sequences of CP and 3''UTR regions was used for constructing a hairpin structure for PVY resistance. This construct was used for Agrobacterium-mediated transformation of tobacco variety Wisconsin 38. Following transformation، 61% of the resulting transgenic plants in T0 were resistant to PVY and the inheritance of resistance to the next generation of nine different lines was confirmed using DAS-ELISA which confirmed the immunity in all tested plants. One of these lines was tested against 12 different PVY isolates، including four Iranian and eight foreign ones، and the immunity against PVY was confirmed based on the lack of symptoms and the results of DAS-ELISA and RT-PCR tests. The results showed that the hairpin construct had a high-performance for generating transgenic tobacco plants with resistance against different PVY strains.

During 2008-2009، a total of 154 gladiolus (Gladiolus grandiflorus) corms were collected from the distribution centers in Karaj (Iran). After cultivation، fresh leaf samples of all germinated corms were tested by DAS-ELISA، which revealed the infection of 74. 02% of plants with Bean yellow mosaic virus (BYMV). Following biological purification of an isolate، designated as BYMV-GPK، its host range was determined. The molecular mass of BYMV-GPK coat protein was estimated as 34 KDa by SDS-PAGE، which was confirmed by western blot analysis. DAS-ELISA and tissue print immunoassay (TPIA) techniques showed similar sensitivity in detection of BYMV in leaf tissue of infected gladiolus plants، but none of them was able to detect BYMV in corm tissue of the same infected plants. Two fragments of GPK genome، corresponding to HC-Pro and 3´ end regions with 600 and 1100 bp in length، respectively، were amplified by RT-PCR. Phylogenetic analysis based on the nucleotide and deduced amino acid sequence of HC-Pro and genomic 3´ end region showed that BYMV isolates clustered into separate groups according to their host origin and GPK isolate was closely related to a trifolium and other gladiolus isolates.

Mixed infection of plants with more than one virus results in different types of interactions. Interaction between two recently identified lettuce infecting viruses in Iran, Lettuce X potexvirus (LeVX) and Rubus Chlorotic Mottle sobemovirus (RuCMV), was studied on co-infected Chenopodium murale and Chenopodium quinoa plants. Plants were mechanically inoculated with either LeVX, RuCMV or both. Co-infected plants of C. quinoa developed much more severe symptoms than the singly infected ones, with enhanced RuCMV accumulation level, as determined by quantitative ELISA and Western blot assay, indicating that the interaction of RuCMV with LeVX is synergistic in C. quinoa. Co-infection of C. murale plants resulted in symptom severity while with no detectable increase in RuCMV accumulation level, indicating that RuCMV interaction with LeVX is not synergistic in this plant. Conversely, LeVX accumulation level in either of C. quinoa or C. murale dually infected plants was significantly reduced, suggesting that LeVX interacts with RuCMV antagonistically in co-infected plants of both Chenopodium species.