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During surveys conducted in several greenhouses in northwestern and southwestern Iran in the fall of 2021-2022, viral symptoms such as leaf mosaic, leaf deformation, and shoestring were observed in several tomato plants. Reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers to detect the tomato brown rugose fruit virus (ToBRFV). A 458 base pair fragment corresponding to a part of the coat protein gene was amplified in all symptomatic samples. Sequence blast analysis and multiple alignment of the sequenced isolate with those of the isolates obtained from GenBank revealed 99.76% nucleotide sequence identity with existing GenBank isolates. Phylogenetic analysis grouped ToBRFV isolates into three clades, the Iranian isolate (Jol-F-2022) fell into clade II. The possibility of mixed infection in the samples with important viruses was investigated using universal orthotospovirus primers, pepper mild mottle virus (PMMoV), tomato mosaic virus (ToMV), and cucumber mosaic virus (CMV)-specific primers. In five samples, CMV-specific primers amplified a 657-base pair fragment of the envelope protein gene, whereas no fragment was amplified with general primers for the orthotospovirus genus and specific primers for PMMoV, ToMV. Nucleotide blast (BLASTn) analysis revealed a nucleotide sequence identity around 95.13-98.85 between the Iranian CMV isolate and the corresponding sequences in the GenBank. Phylogenetic analysis based on the nucleotide sequences of the coat protein gene grouped the CMV isolates into three major groups, the Iranian isolate grouped with the Indian CMV isolates in the subgroup IB. Biological studies were performed by mechanical inoculation of an infected sample on Nicotiana rustica. The inoculated plants showed severe mosaic symptoms at 15 days post inoculation (dpi), and infection by both viruses was confirmed through RT-PCR using specific primers. This study presents the first report of mixed infection of tomato plants with ToBRFV and CMV in Iran.

The Prunus necrotic ring spot virus (PNRSV) poses a significant threat to the global stone fruit industry. In this study, samples suspected of PNRSV infection were gathered from Iran’s primary stone fruit-growing areas. The coat protein (CP) gene of the isolates was amplified, and the nucleic acid and amino acid sequences of the PNRSVs were determined. Phylogenetic analysis revealed that these isolates belonged to the PV-96-II, a prominent group of PNRSVs found worldwide. An isolate from this clade was chosen to express the CP gene in Escherichia coli. The CP gene of this isolate was cloned into the pET28 vector for expression, and the resulting coat protein was purified as a native protein using Ni-NTA agarose. The purified protein served as a recombinant antigen to generate anti-PNRSV-CP antiserum in rabbits. Purified anti-PNRSV-CP IgG and conjugated IgG showed specificity and sensitivity, successfully detecting expressed CP and PNRSV isolates in infected stone fruit trees through various serological and sero-molecular techniques such as plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), Double Antibody Sandwich-ELISA (DAS-ELISA), and western blotting. These antibodies can be valuable in virus and plant screening studies, as well as serological and sero-molecular tests. This study represents the first documentation of a polyclonal antibody preparation against the CP of an Iranian PNRSV isolate, offering potential assistance in controlling and preventing this economically significant virus in the future.

Goal: 

production of antibodies against the coat protein of Apple stem grooving virus and their application in diagnostic assays and biosensor design represents a crucial strategy for effective plant virus detection. ASGV is a significant pathogen affecting pome fruit trees. The objective of this study was to express the coat protein gene of ASGV in E. coli, generate antibodies against the expressed protein, and assess their effectiveness in diagnostic assays.

In the PCR, we successfully amplified a 714 bp fragment corresponding to the complete coding region of the ASGV coat protein gene. The amplified fragment was then cloned into the pTG19 vector. Subsequently, the desired fragment was subcloned into the pET28a (+) expression vector using BamHI and EcoRI restriction enzymes. The resulting construct was then introduced into E. coli BL21(DE3) cells for gene expression. To determine the optimal conditions for protein expression, the transformed cells were induced with one mM IPTG for various durations (3, 4, 6, and 16 hours). The expression of the protein was confirmed through SDS-PAGE electrophoresis and western blot analysis. Following the purification of the expressed protein, it was utilized as an antigen for immunizing rabbits. Immunoglobulins (IgGs) were subsequently purified from the rabbit serum, and some of them were employed for conjugate production. The efficacy of the conjugates was evaluated in serological assays.

Sequencing analysis of the amplified fragments conclusively verified their correspondence to the ASGV coat protein gene. Moreover, PCR and enzymatic digestion of the resulting clone provided further evidence of the successful construction of pET28-ASGV-CP. Comprehensive sequence analysis of the constructed plasmid in both directions confirmed the accurate insertion of the coat protein gene into the expression vector, while also confirming the absence of any nucleotide mutations. Following optimization, the expression of the ASGV coat protein was successfully achieved, resulting in an approximate size of 27 kDa. This confirmation was attained through SDS-PAGE electrophoresis and subsequent western blotting, conducted four hours after induction. The direct, indirect, and dot-blot assays were performed to assess the efficiency of the conjugate. The results indicated that the optimal concentration of the conjugate was approximately 1:1000.

The generated and conjugated antibodies exhibit suitable titers relative to the antigen concentration and demonstrate specificity and effectiveness in the detection and identification of Apple stem grooving virus (ASGV). These antibodies can be utilized for various serological diagnostic kits, including ELISA, offering valuable applications in virus detection and identification.

Tomato bushy stunt virus (TBSV) is one of the important tomato viruses in Iran and all over the world. In this study, the effect of mycorrhizae in Tomato bushy stunt virus infection was investigated. For this purpose inoculum of mycorrhizal fungus Rhizoglomus irregulare was added to the plant growing medium at the time of tomato transplanting and after four weeks, TBSV was inoculated mechanically to the plants. The rate of virus multiplication was evaluated by Real-time PCR at time intervals of 19, 25 and 31 days after inoculation. The treatments included control plants (C), plants infected with TBSV (V), mycorrhizal plants (M) and mycorrhizal plants infected with TBSV (MV). The results of investigating the amount of virus accumulation in plants showed its decrease in MV plants compared to V plants, and this decrease was more obvious in more advanced stages of the disease. Investigating the expression level of AGO2 and MYB33 genes also revealed that these genes had a significant increase in MV plants compared to V plants. This increase indicates an increase in the resistance of MV plants to the virus compared to V plants and it also confirms the lower reproduction of the virus in tomato plants inoculated with mycorrhiza. The results of this research show that the use of mycorrhizal fungi as a stimulant of the plant's defense system may be a good way to defend against this viral disease.

Global warming is an important phenomenon with many effects on the environment, which is associated with an increase in greenhouse gases such as carbon dioxide, methane and other gases. Accumulation of greenhouse gases, increase or decrease in temperature, and change in rainfall all affect the distribution of plant pathogens and the incidence of disease. On the other hand, by affecting the carriers of pathogens, climate change can cause them to disperse in a wide area or cause pathogens to enter new areas. On the other hand, the effect of global warming on plants is different and diverse depending on the plant species and its pathogens and according to different environmental conditions. These effects can be harmful, positive or neutral. However, according to the pathogenicity triangle, one of the central mainstays of the disease rate is environmental conditions; therefore, plant diseases are strongly affected by the environment; if a sensitive host is placed in unfavorable conditions, it will not be infected by an invasive pathogen. Based on the conducted research, temperature changes in the environment cause the emergence of new diseases and the spread or limitation of diseases. This article discusses the essential effects of global warming on the occurrence and spread of plant diseases in new areas.

Apple chlorotic leaf spot virus (ACLSV) is one of the most important viruses infecting fruit trees. Preparation antibodies, ELISA kit and, biosensors are one of the applicable strategies to rapid and efficient screen a number of viral samples. To aim, the recent study was conducted to raise recombinant polyclonal antibodies to rapid detection of Apple chlorotic leaf spot virus by expressing coat protein gene in the prokaryotic system. First, the pTG19-ACLSV-CP was transformed to E. coli DH5α, the replicated plasmids were extracted and double digested was optimized by restriction enzymes, then the released fragment (Coat protein gene) was cloned into pET28 as an expression vector. Next, the expression construct (pET28a-ACLSV-CP) was transformed into E. coli strain BL21 (DE3) to express the coat protein gene. After optimization of expression the transformed cells, a protein band an approximate size of 27 kDa (22 kDa for coat protein and about 5 kDa for histidine tags) was observed at four hours after induction. The recombinant protein was purified by native methods using Ni-NTA Agarose column, then purified proteins were injected into New Zealand female rabbits in four steps as antigen. IgGs were purified from serums raised from immunized rabbits using an IgG purification kit. The efficiency of purified IgGs was approved that a 1: 1000 concentration of recombinant anti-ACLSV-CP antibodies are efficient to detect the desired antigens. The results showed the raised antibodies have enough specificity to detect Apple chlorotic leaf spot virus in gardens and seedlings.

Tomato brown rugose fruit virus (ToBRFV) is an emerging pathogen that generally causes disease in greenhouses. This virus causes yellow areas, malformation and brown necrotic lesions on tomato fruits. This virus, which has a global distribution, has recently been reported using molecular methods in the greenhouses of few provinces of Iran. According to the report of this virus from Iran, the characteristics of the ToBRFV, its symptoms, host range, mode of transmission and spread, and its management methods have been described based on previous studies.

Apple stem grooving virus (ASGV) is one of the important viruses infecting apple trees. In this research, 87 symptomatic and symptomless leaf and bark samples were collected from the apple orchards in west and south west of Zanjan and east of Kurdistan provinces to detect ASGV. After total RNA extraction from 50 out of 87 samples, cDNAs were synthesized by reverse primer and PCR optimized using a pair of specific primer corresponding to complete coat protein gene. A DNA segment with 728 bp length corresponding to complete coat protein gene was amplified from twelve samples by RT-PCR. Nine PCR-products including four samples cloned into pTG19 and five samples without cloning were sequenced. The sequencing and blast results revealed that the fragments belong to coat protein gene of Apple stem grooving virus. Phylogenetic analysis of new Iranian isolates and those retrieved from GenBank by MEGA 7 and Neighbor-Joining methods in Jukes-Cantor model showed that the new Iranian isolates were placed in a sub-clade with reported Iranian, Indian, South Korean and Chinese isolates. The result revealed that the least dN/dS ratio belonging to Iranian isolates and the most gene flow was calculated between isolates from Iran, China and South Korean. This is the first report of Apple stem grooving virus from the Zanjan region.

Tomato spotted wilt virus disease is one of the most important tomato diseases that causes damage in the country. To identify inbred resistant lines against Tomato spotted wilt virus (TSWV) in the present study, 64 tomato genotypes including 28 tomato hybrid (F1) cultivars were initially evaluated for the presence of Sw-5b conferring resistance using a codominant SCAR (Sequence characterized amplified region) molecular marker, NCSw003. Based on the results obtained from this stage of the study, only four tomato hybrid cultivars namely Sereen, Brivio, Jam and Xaman, carried the resistance allele. Based on the results of the second stage and 240 selected F6 inbred lines with appropriate agronomical traits those were generated from four the above-mentioned heterozygous cultivars, The inbred lines generated from Sereen, Jam, Xaman and Brivio carried out 35.6, 35.1, 25.7 and 22.2 percent homozygous resistant allele specific, respectively. Furthermore, single nucleotide polymorphism in Sw-5b gene allele was investigated for F6 selected progenies of three heterozygous cultivars, Sereen, Xaman, and Brivio using high resolution melting (HRM) technique. The comparison between the results of screening by HRM and NCSw-003 codominant SCAR marker showed 86, 98.6 and 91 consistencies, respectively. Considering identical results of both markers used for each of the above cultivars, 21, 18 and 12 pure homozygous resistant lines were identified, respectively. Also, the results of the study using NCSw-003 marker alone showed that 20 pure lines obtained from Jam cultivar have dominant homozygous resistance. The identified resistance lines with homozygous resistance can be used after biological evaluation as a resistant parent in tomato breeding programs and introduction of hybrid cultivar.

 Viroids are classified in two families including Avsunviroid that consists of three genera (Pelamoviroid, Avsunviroid and Elaviroid) and pospiviroid including five genera (Apscaviroid, Hostuviroid, Cocaviroid, Coleviroid and Pospiviroid). Grapevine-infecting viroids belong to the three genera in the pospiviroid family. A total of 121 symptomatic and non- symptomatic leaf and green shoot samples were collected from the main vineyards in Zanjan province to detect the important viroids infecting grapevines. Total RNAs were extracted from the samples and then cDNAs were synthesized by cDNA synthesize Kit with a random hexamer primer. Specific primers used to detect Grapevine yellow speckle viroid 1(GYSVd-1), (GYSVd-2) Grapevine yellow speckle viroid 2, (HSVd) Hop stunt viroid, Australian grapevine viroid (ASGVd) in reverse transcription polymerase chain reaction (RT-PCR). The results revealed that the specific DNA fragments corresponding to each viroids were amplified in PCR. Percentage of infections were 14%, 16%, 20% and 22% for GYSVd-1, GYSVD-2, HSVd and ASGVd relative to total number of examined samples, respectively. Sequencing of seven selected PCR-amplified fragments showed that each newly sequenced segment was classified in a clade with the previously reported respective isolates from Iran. In addition, the most mixed infection related to HSVd and other viroids. This is the first assay to detect the four important viroids infecting vineyards form Zanjan province.

Biochar is a coal that is prepared during the pyrolysis process by burning plant biomass and agricultural waste in the presence or absence of oxygen at a temperature around 450-550°C that is used as a fertilizer. In view of agriculture, one of the benefits of biochar is waste management in agriculture. This solid material is rich in carbon and has a large capacity to reduce greenhouse gases such as carbon dioxide and methane released from waste due to very slow decomposition rate compared to other organic materials, and can keep carbon for a thousands of years. Biochar is widely known as an efficient way for carbon sequestration and increasing soil fertility. It is used as a soil texture improver or to regenerate lands as well as protecting the environment against certain pollutants. By the addition of biochar, the stability of soil will increase the organic carbon against environmental factors and reduces mineralization which increases organic carbon and improves soil structure. Increasing water holding capacity and activity of microorganisms and better absorption of nutrients from soil is one of the main roles of biochar. In addition, biochar will improve crop yields, management of agricultural waste. Increasing plant's systemic resistance to a number of plant pathogens and uptake of pesticide and herbicide residues from the soil will cause the growth of healthier plants. Development of organic agriculture and problems that resulted in air pollution has made use of this type of fertilizer around the world, however, this organic material has remained somewhat unknown in Iran.

Plant viruses are important because of their threat to produce of crops products and also their application as research tools in molecular plant pathology and other fields in biotechnology is important. The most research and achivments in plant pathology depends on ability of plantchr('39')s viral genomes to preserve, replicate and modify as infectious clones. The infectious clone is a full-length DNA clone in which infectious transcripts can be obtained in vitro or in vivo conditions with a suitable promoter. Infectious clones can be used to study the life cycle of viruses, replicate and express of viral genomes, understanding of virus-host interaction, functional genomics and germplasm screening as a part of pre-replication programs for virus resistance. However, the infectious clone of plant viruses may pose a risk to the environment due to its ability to return to the wild-type mode through recombination. This scientific review introduces the infectious clone in plant viruses and surveys the advantages and disadvantages of working with genetically modified plant viruses.

With progress in science, new technologies open a window to answer the questions. Besides biological, serological and molecular methods, Next-generation sequencing (NGS) is one of the methods that changed our understanding of plant viruses, especially in the areas of identification, genome and transcriptome sequencing, evolution, taxonomy and quarantine programs. Recent methods make it possible to detect and identify all known and unknown viruses in a plant without the need for prior knowledge of the pathogen by the identify of total viral content and transcriptome of plant samples in healthy and disease conditions whearse ther are problems such as storage and analysis and interpretation of a large amount of data produced. Increases in the speed of detection of new viruses, viroids and hosts can prevent entering new viruses and viroids from foreign countries. More accurate and faster identification of plant viruses and their full effects on different hosts is one of the most important steps in disease management. In this article, we will review the applications of NGS in the diagnosis and detection of viruses, identification of diseases of unknown etiology and the study of host-pathogen interactions.

Cucurbit crops belonging to the family Cucurbitaceae are one the most important horticultural crops worldwide and cultivated widely in Iran. To aim detection and phylogenetic analysis of potyvirus members, during 2015-2016, 215 leaf samples from cucumber, melon, watermelon, and squash symptomatic with deforming and reduction in leaf size, blistering, mild and severe mosaic, fruit deformation, and stunting were collected from 10 major cucurbits-growing locations in West Azerbaijan province. Following mechanical inoculation of Cucurbita pepo as an indicator plant with symptomatic cucurbit crop extracts, a variety of symptoms developed on the plants. Then, total RNA extracted from 70 symptomatic leaf samples, and partial RNA-dependent RNA polymerase (NIb) (350 bp) was amplified using universal primer pairs (NIb2F/NIb3R) by reverse transcription-polymerase chain reaction (RT-PCR). The infection by potyviruses in 38.75% of the samples was confirmed based on the result. The five PCR products belonging to different hosts at the expected size (350bp) were sequenced. One sample (Ir-Na: MH491979) was determined to be infected by the Watermelon mosaic virus (WMV) whereas four samples (Ir-Ma:MH491979, Ir-Ng:MH491980, Ir-Pi:MH491981, and Ir-Ur:MH491982) infected by Zucchini yellow mosaic virus (ZYMV). In the phylogenetic analysis based on Maximum-likelihood, three ZYMV isolates (Ir-Ng, Ir-Pi, and Ir-Ur) were placed in subgroup I whereas one isolate (Ir-Ma) was placed in subgroup II belonging to group A. Also, WMV isolate (Ir- Na) was placed in CL phylogroup.

In this study the infection of common bean fields by Bean yellow mosaic virus, BYMV in some important regions of Zanjan province was investigated and phylogenetic and population relationship of Iranian BYMV isolates with other isolates from GenBank was studied. To detect BYMV from bean fields, 156 leaf samples were collected and divided in different groups based on symptoms and regions. Finally, 81 leaf samples were selected for total RNA extraction and BYMV infection assay by RT-PCR. Then, a specific primer pairs was applied to amplify 900 bp of coat protein gene using RT-PCR. Nine samples showed infection with BYMV and two representative amplified fragments were sequenced. Phylogenetic analysis was performed using MEGA 7 software and population parameters including recombination prediction, mutations and genetic diversity were calculated by RDP4, DnaSP and SDT softwares. The results of phylogenetic analysis showed that the newly detected isolates were grouped in a cluster along with isolates from the South Korea, the USA and Australia that have been reported from different hosts. In addition, the most genetic distance was calculated among Iranian isolates and isolates from Australia and Japan. The results showed that the highest ratio of non-synonymous to synonymous mutations (dN/dS) is related to the Clade 1 and the highest nucleotide diversity and the lowest π is related to this group including Iranian isolates. In other sub-clades, there were correlation between isolates and geographical regions.

Tomato bushy stunt virus (TBSV), a member of the genus Tombusvirus is one of the causal agents for curly disease in tomato plants. In this study, the interaction between a mycorrhizal fungus, Rhizoglomus irregular, and TBSV in tomato plants was investigated. In a completely randomized design experiment, tomato seedlings were inoculated with R. irregular and after four weeks they were inoculated with TBSV. Four treatments were included: control plants (C), TBSV -infected plants (V), mycorrhizal plants (M), TBSV -infected mycorrhizal plants (MV). Nineteen days after inoculation the infected plants were tested for symptom production and virus accumulation. Results of symptoms evaluation based on the disease severity index showed a lower disease severity in MV plants compared with V plants. Supporting this result, a lower level of virus accumulation was observed in V plants which was more significant at long-term infection. The expression of methylation-related genes including ADK, HEN1 and MET1 was tested by Real-time PCR. Results showed that the expression of these genes was significantly higher in MV plants as compared with V plants. An increase in the expression of methylation-related genes in MV plants indicates that resistance to the virus is likely to occur through methylation and also supports the lower level of virus accumulation in MV plants.

Cucumber mosaic virus (CMV) is a type member of Cucumovirus genus in the family Bromoviridae. In this research, the expression profile of several genes related to development and methylation was tested in a sensitive variety of tomato infected by CMV-Fny. Tomato inoculation was carried out using a standard isolates of CMV (Fny). To this aim, transcripts of RNA1, RNA2 and RNA3 of CMV were transcribed and then, inoculated using sap inoculation on tobacco plants at the four-leaf stage. After symptoms induction, leaf samples were collected at 7, 14 and 21 days post inoculation (dpi). The virus infection was confirmed by RT-PCR using CMV-CP specific primers corresponding coat protein gene. The expression of several genes related to development and methylation including Hsp90, AGO1 and AGO4 was tested by real-time PCR. The results showed that the expression of HSP90 was decreased after infection by CMV compared to healthy plans. In addition, the expression of AGO1 was increased at 7 and 14 dpi whereas the expression was decreased compared to healthy plans at 21 dpi. The expression of AGO4 which has the role in plant defense was increased in infected tomato plants at 14 and 21 dpi and AGO4 expression was decreased compared to healthy plant in 7 dpi. It seems, changes in AGOs expression refer to late induction of plant resistance against the virus infection.

In spite of existing of several methods for detection of viruses, time consuming is major limitation of them. So, developing faster and real time method is important to certificate the plant materials in quarantine stations and virus management. In the recent research, nanotechnology was employed to detect Potato virus S (PVS) using gold nanoparticles (AuNPs). Colloidal AuNPs were prepared through citrate reduction and conjugated to a specific antibody against PVS coat protein. Conjugation was optimized by changing the pH of AuNPs and antibody concentration. Antibody coated nanoparticles were used to detect different concentrations of antigen using spectrophotometry. After binding the antibody to the gold nanoparticles which cause an increase in particle size, the SPR peak was displaced in range of 4-5 nm and shifted from 524 to 530 nm. The binding of the antigen to the nanoparticle also caused a change in the particle size and an increase maximum absorbance from 530 to 539 nm. No spectral change were seen when PVY and CMV used as controls. This study is the first attempt of nanoparticle usage in quick and easy detection of PVS

The Apple chlorotic leafspot virus (ACLSV) is among of the most common virus infecting pome and stone fruit trees around the world. The virus has spread throughout the world and has been reported in many hosts such as; apples, pears, peaches and cherries. The purpose of this study is to express the coat protein (CP) of ACLSV in E. coli. CP expression in E. coli has been developed to prepare antigen for antibody production without needs to purify the virus from infected plant. In this study, complete CP gene (582 bp) from an isolate of ACLSV (SSa) was amplified by specific primers, MF and MR. The amplified CP gene was ligated to TA cloning vector (pTG19-ACLSV CP) and transformed into E. coli strain DH5α. Transformed cells were selected on LB containing ampicillin, X-Gal and IPTG, and recombinant plasmids were confirmed by restriction analysis. Then, the pTG-ACLSV CP was sub-cloned into pET28a (+) as expression vector that digested by BamHl and XhoI restriction enzymes. Finally, the pET28a-ACLSV CP was transformed into E. coli strain BL21 by heat shock method. For ACLSV CP expression, the cells containing pET28a-ACLSV CP were induced by 1 mM of IPTG and the protein was extracted two and four hours after induction and analyzed in SDS-PAGE. The SDS-PAGE result showed that ACLSV CP have been expressed that is expected based on additional tags from plasmid.

Gene expression in bacteria have already been used to study the protein of viruses and to produce specific antibodies. In this research, potato samples were collected from eight provinces of Iran and were tested for Potato virus S, as one of the most destructive viruses of potato fields, by DAS-ELISA and RT-PCR. Dominant isolate of PVS was selected according to coat protein (CP) gene sequences following phylogenetic analysis. Full length CP gene was amplified, cloned and sequenced, then was digested from pJET1.2/blunt vector, ligated into the expression vector pET-28a and the construct (pET-28a:PVS:CP) was transformed into Escherichia coli BL21 strain. Gene expression was optimized by induction with 0.5, 1 and 2 mM final concentrations of IPTG for 3, 4 and 6 h and was verified by SDS-PAGE and Western blotting. Based on the nucleotide analysis, the Iranian isolates of PVS were divided into three groups that one group with 22 members known as the dominant group. The expression of recombinant CP of about 34 kDa was proved by western blotting. Induction by 1 mM IPTG for 4 h proved to be the most efficient method of expression. This is the first report of the expression of PVS CP gene, which is important for the preparation of anti-PVS antibody.

To determine incidence of nepoviruses in vineyards of zanjan province, 168 symptomatic and non-symptomatic samples were collected during season growing 2014-2015. Total RNA was extracted from the 57 leaves and green shoots of selected samples and then cDNAs were synthesized. Genomic segment of Arabis mosaic virus (ArMV), Grapevine deformation virus (GDeV), Grapevine fan leaf virus (GFLV) and Tomato ring spot virus (ToRSV) was amplified by polymerase chain reaction (PCR) using specific primers. The result revealed that the expected bands were amplified at 15.7, 15.7, 10.5 and 14% of different samples belong to ArMV, GDeV, GFLV and ToRSV, respectively. Following sequencing and multiple alignment of nucleotide sequence of amplified fragments with isolates that retrieved from NCBI. Phylogenetic tree was created by MEGA6 software using Neighbour-Joining method. The result revealed that the Iranian isolates were grouped with reported isolates from different regions around the world based on geographical. This is the first comprehensive study based on molecular methods to determine grapevine nepoviruses in Zanjan province and the first report of GDeV from Zanjna based on our knowledge.

Infectious diseases account for more than 45% of total deaths in developing countries. Vaccination is the most effective means to prevent infectious diseases but current vaccine production methods are complex and expensive in technology. Such conditions make it impossible for a large part of the developing and poor countries to have access to vaccination. New strategies for producing vaccine subunits include the use of different systems, such as fermentation, bacteria and mammalian cells. But these systems have limitations, including development costs, Transportation and cooling systems, making them impractical. For this purpose, plant - derived vaccines are considered as a new perspective on the production of vaccines. In this method, small fragments of DNA encoding the epitope or antigen to be linked to the coat protein gene of the plant viruses. Then the recombinant virus is used to infect the plants. In this way, a wide range of plants have been used to produce vaccines against diseases. In this article, we describe the methods of producing oral vaccine using transgenic plants and the advantages and disadvantages of this technology.

Because of limitations in antibody production against plant viruses, coat protein expression has been developed to prepare antigen for antibody production. In the recent research, Nucleocapsid gene of Tomato spotted wilt virus was amplified from a local strain in Iran. Cloned segment in TA vector (pTG19TSWV-N) was sub-cloned into pET32a as expression vector that digested by XhoI and BamHI. Then, pET32TSWV-N was transformed in two different strains of E. coli BL21(DE3) and BL21(DE3) pLysS by heat shock method. For protein expression, the transformed cells were induced by 1mM of IPTG in both strains. The protein was extracted four hours after induction and analyzed in SDS-PAGE. The SDS-PAGE result showed that a 48 kDa protein have been expressed that is expected based on additional tags from plasmid. The result revealed that nucleocapsid had been expressed in both strains whereas protein expression was little more in BL21(DE3) pLysS.

Among several diseases of gapaevine, fan-leaf disese is the most imprtant viral diseases in the garapevine orchards around the world and Iran. Furthermore, early detection and diagnosis of the disease and screning the infected plant could be useful to disase management. In the recent research, local GFLV isolate was selected to prepare the recombinant antibody to diagnosis the infected samples. In the recent research, after the rabbit immunization by coat protein and movement protain of GFLV that expressed into E.coli, the rabbits were bled and the serum fractions were collected and stored at -20°C until required. After the titration of serum fraction, the immunoglobulin G (IgG) from the antiserum was purified using DEAE cellulose column and the efficiency of purification was estimated by the light absorption at 280 nm (A280) and electrophoresis on 12% gel SDS-PAGE. After the IgG purification, indirect-ELISA was done by different dilution including 1:500, 1:1000 and 1:2000. The result showed, IgG-CP-GFLV revelaed the best absorbance in 1:500 and 1:1000 IgG-cp diluted wherase the IgG-MP-GFLV showed the best reaction in 1:500 IgG-MP dilution. DIBA by purified IgGs showed that the IgGs could be detect the related antigenes in 1:500 dilution fot both. Finally, the result revealed that IgGs purified by DEAE cellulose column could be applied in serological and seromolecular test.