rt-pcr

Grapevines, as a vital agricultural product, face significant challenges from pests and diseases that adversely affect both the quality and quantity of the crop. Among these, viruses are particularly detrimental, with 86 different species identified in grapevines. The viruses associated with grapevine leafroll disease (GLD) are considered the most prevalent in vineyards. Specifically, six species of grapevine leafroll-associated viruses (GLRaVs), belonging to the family Closteroviridae, are linked to this disease. These viruses typically enter vineyards through infected cuttings and are disseminated by vectors, notably mealybugs. The symptoms of GLD manifest as downward rolling of leaf margins, leaf interveinal reddening, and leaf interveinal chlorosis across various grapevine varieties. These symptoms negatively impact the quality of the berries, ultimately affecting yield and marketability. The GLRaV-1 genome is composed of positive-strand RNA and contains ten open reading frames (ORFs), which play crucial roles in its biology and pathogenicity. In Iran, GLRaV-1 has been identified as one of the most widespread grapevine viruses, exhibiting high genetic diversity that complicates management and control efforts. This study focuses on the identification of GLRaV-1 and the phylogenetic analysis of its isolates in the vineyards of Razavi Khorasan province. By understanding the genetic relationships and variability of GLRaV-1 isolates, this research aims to provide insights which could be useful for designing effective management strategies and improving the overall health of grapevine in the region.

In the spring of 2020, a study was conducted in the Kashmar region of Khorasan Razavi province, focusing on grapevine plants exhibiting distinct symptoms of viral infection. A total of 70 samples were collected from affected plants. The RNA was extracted from these samples to facilitate the detection of grapevine leafroll-associated virus 1 (GLRaV-1). The initial identification process involved reverse transcription-polymerase chain reaction (RT-PCR). This method confirmed the presence of GLRaV-1 in 22 samples, marking a crucial step in understanding the viral infection's prevalence. To ensure the accuracy of the RT-PCR results, Sanger sequencing was used on the amplified fragments from three selected samples. This technique provided definitive confirmation of the viral identity by analyzing the nucleotide sequences, thereby validating the initial findings from the RT-PCR. Data analysis included several critical components. Nucleotide sequence similarity analysis was performed to compare the identified GLRaV-1 isolates against known sequences in databases, assessing their similarity and identifying potential genetic variants. Following this, multiple sequence alignments were conducted using MAFFT software, arranging the sequences to highlight similarities and differences among the isolates. The phylogenetic tree was constructed using the Maximum-Likelihood method with MEGA7 software.

The samples collected from black grape vineyards in Kashmar exhibited mild yellowing and downward rolling of leaf margins, symptoms commonly associated with increased anthocyanin levels in red grape varieties. In samples infected with GLRaV-1, a fragment of the minor capsid protein 2 (Cpm2) gene was successfully amplified, while no such fragments were detected in healthy plants. This indicates the presence of GLRaV-1, a virus recognized as one of the common pathogens affecting grapes across various regions of Iran. The GLRaV-1 is primarily transmitted through vectors or contaminated cuttings, which emphasizes the importance of monitoring and controlling these pathways to prevent outbreaks. Sequence comparisons revealed that nucleotide identity among Iranian isolates ranged from 84.2% to 100%, while identity with GenBank isolates varied between 77.6% and 87.7%. This high level of genetic diversity among the isolates may enhance the virus's adaptability to different grape varieties, posing challenges for management and control. The observed diversity at the genomic level was consistent with previously reported data regarding the sequence divergence between the two GLRaV-1 groups (Alabi et al., 2011; Sabella et al., 2018; Fan et al., 2015). Phylogenetic analysis further demonstrated that Iranian isolates of GLRaV-1 clustered into two main groups, indicating that the phylogenetic relationships are not strictly related to geographical origin. In previous studies, global isolates of GLRaV-1 have also been distinguished into two different groups based on the gene encoding the minor coat protein 2 (Alabi et al., 2011; Elci, 2019). This finding suggests that the spread of the virus may occur independently of the location, highlighting the need for comprehensive management strategies. To mitigate the spread of GLRaV-1, the use of virus-free plant materials is essential (Alabi et al., 2011; Bruisson et al., 2017). The genetic diversity of GLRaV-1 significantly influences the epidemiology of grapevine leafroll disease, underscoring the importance of producing virus-free plants. Continued research is crucial to understand the underlying causes and effects of this virus. Additionally, developing more sensitive and specific diagnostic tests, along with new control strategies, is vital for effectively managing GLRaV-1 and safeguarding grapevine health.

Hippeastrum/amaryllis (Hippeastrum hybridum Hort.) belongs to Amaryllidaceae. Plants of the hippeastrum exhibiting virus-like symptoms of chlorotic ring spots and concentric oval patterns were found in the collection of ornamental geophytes of the Ornamental Plants Research Center, Mahallat, Iran. Amplification of the genome using general primers of the Orthotospovirus genus, and specific primers of capsicum chlorosis virus (CaCV) led to the amplification of the expected fragments in some of the collected samples. Sequencing of amplified fragments proved the infection of some of the samples with CaCV and iris yellow spot virus (IYSV).No simultaneous infection with these two viruses was observed in any of the samples. This is the first report of the occurrence of members of the genus Orthotospovirus on the hippeastrum plant from Iran.

Grapevine virus A (GVA) belongs to Vitivirus genus and is one of the most important causes of wood rugose in grapes. During the years 2019 to 2021, a total of 79 leaf samples showing symptoms of leaf yellowing and reddening from vineyards of Khorasan Razavi Province (including Bardaskan, Kashmar, Khalilabad, and Mohammadiyeh) were collected. The initial infection of the samples to GVA was directly checked using an ELISA test, and the final confirmation of results was performed by RT-PCR with specific primers. Total RNA extraction was performed from scraped bark of young stems using CTAB method. GVA was detected in 14 samples through the ELISA test, and a fragment of 238 base pairs from the p10 protein was amplified in these 14 samples using RT-PCR. The amplified products from four samples were ligated into the pTG19-T vector, and the recombinant plasmids were sequenced bidirectionally. The obtained sequences were compared with the other GVA sequences in GenBank using BlastN tool and the nucleotide similarity was 85.7-95%. The phylogenetic tree based on p10 protein showed there are four main groups, and Khorasan Razavi isolates classified within the fourth group. Comparison of p10-protein sequence of Iranian isolates with other isolates showed there is a conserved region in N-terminal region, which includes an arginine-rich motif followed by a zinc-finger motif. There have been many studies on other viruses of grape in this province, but this is the first study on ithe phylogenyof GVA isolates in the vineyards of Khorasan Razavi province.

Beet necrotic yellow vein virus (BNYVV) and Beet black scorch virus (BBSV) have been observed in infected sugar beet roots from Iranian fields. This study aimed to detect the two viruses simultaneously to save time and energy during the infection detection process.

Three methods were utilized in this study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect two viruses following total RNA extraction from the farm sample. In the first method (the standard method), the mentioned viruses were identified by amplifying 545 bp fragments from the TGB region of the RNA2 genome of the BNYVV virus and 305 bp fragments from the 3'UTR region of the BBSV virus using specific primers. Viruses on an infected root were identified using RT-PCR with primers specific to each virus and performed independently. In the second and third methods, we employed simplex and duplex RT-PCR gradient methods to simultaneously identify two viruses at annealing temperatures of 52, 54, and 56 °C. To this end, virus-specific primers (designed from a part of the gene encoding the coat protein) were used separately but simultaneously with a common PCR program in a thermocycler. These specific primers amplified 391 and 453 bp fragments of the BNYVV and BBSV coat proteins, respectively.

The study's findings show that by performing simplex and duplexRT-PCR simultaneously and optimizing the annealing temperature of primers from the gene encoding the protein coat, target fragments of 453 and 391 bp from BBSV and BNYVV, respectively, are amplified. This study found that an annealing temperature of 56 °C is the optimal temperature for duplex RT-PCR to separate the two viruses. Consequently, simultaneous identification of the BNYVV and BBSV was achieved through this method.

The present study demonstrates that simplex and duplexRT-PCR are suitable for simultaneous detection and are cost-effective in terms of saving time and energy. They can also be used to prepare distribution maps in sugar beet cultivation areas and to evaluate and identify resistant sugar beet lines in breeding programs under greenhouse conditions.

Barley yellow dwarf disease is one of the most prevalent and economically important virus disease of cereals. Barley yellow dwarf virus, BYDV belongs to the family Luteoviridae is causal agent of barely yellow dwarf disease which can result up to 80% yield reduction in wheat. BYDV is present in most regions of Iran. For detection and study on distribution of BYDV, wheat and barley plants showing yellows and dwarfing symptoms were collected from fields of Malayer, Nahavand and Tuyserkan during growing season of 2020-2021. The samples were checked for BYDV infection using indirect-ELISA. The results showed presence of BYDV in most samples. Positive reactions to BYDV antibody in 80% of the Malayer samples, 90% of the Nahavand samples and 80% of the Tuyserkan samples were recorded. Also, total RNA extraction and RT-PCR were performed using specific BYDV-PAV primers; the most prevalent strain of BYDV in most regions of Iran. To verify the results, a sample with symptoms from Tuyserkan selected for sequencing. The BYDV isolate of Tuyserkan had 97% of homology with BYDV isolates were reported from Kahnouj, Karaj, Yazd and Arak, while it showed the least similarity with other Iranian isolates (about 85%).

Barley yellow dwarf virus (BYDV) causes a significant yield reduction and crop loss in cereal and grasses worldwide. Studies revealed that barley yellow dwarf virus is present in most regions of Iran. Wheat and barley plants showing the infection symptoms were collected from cereal fields in Hamedan Province during the springs of 2020 and 2021. Samples were tested for BYDV infection by Indirect ELISA. Positive reactions to BYDV antibody in most samples were detected. After total RNA extraction, RT-PCR was performed to amplify polymerase gene using specific BYDV-PAV primers. BYDV-PAV is the most prevalent species of BYDVs in Iran. Results confirmed BYDV infection. For molecular and phylogenetic comparison, two samples from Hamedan and Asadabad were selected and their polymerase genes were sequenced. The sequencing results revealed that Hamedan isolate had maximum homology with Kahnouj, Karaj, Yazd and Arak isolates (with 95% - 96% identity). The isolates in this group proposed as a distinct species due to their low similarity with other isolates in GenBank. Asadabad isolate had maximum homology with Estonia and America BYDV-PAS with more than 92% homology. The results of phylogenetic analysis revealed that Iranian isolates of BYDV clustered in three distinct groups which shows the presence of a high diversity in the polymerase gene of this virus. This study is the first report of BYDV-PAS species in Iran.

In recent years symptoms of viroid diseases, include stem pitting and gumming have highly incidenced in citrus orchards of Mazandaran. This study was conducted for determining the biological characteristics of Citrus cachexia viroid strains in local tangerine orchards. 150 samples were collected from three important infected districts. Purification was carried out in CF-11 column. Presence of viroid was confirmed with RT-PCR using specific primers. For differentiation of Cachexia viroid isolates, 13 types of citrus were bark grafted on rootstock and symptoms were observed. RT-PCR data showed that 6% of samples were infected only with cachexia, while other samples showed mix infection with exocortis, tristeza and psorosis. According to the assay, the best indicator plant is the Mexican lime (Citrus aurantifolia. Swing.), which could express the typical symptoms of mild to severe infection. Also it is possible to separate severe strains from mild strains based on the symptoms on the local tangerine trees and gumming on the branches can be the primary basis for differentiation.

Grapevine fanleaf virus (GFLV) is a major grapevine infecting virus in the world.

Grapes showing GFLV signs were sampled from the suburbs of the city of Khorramabad in Lorestan Province of Iran in spring 2020 and RT-PCR test was performed to amplify their GFLV-CP gene and the product was sequenced.

Specific primers were able to amplify a 1515 bp fragment of the CP gene. Based on the nucleotide sequence of this fragment, GFLV was first identified in this region. The nucleotide sequence similarity of this isolate was detected at 89.14-95.64% with other isolates in the NCBI library. Also, the phylogenetic tree of these isolates, based on the genomic CP region, grouped GFLV isolates into two groups I and II. The Lorestan GFLV isolate was placed in a subgroup in Group I together with GFLV isolates from the northwestern part of Iran and the Takestan isolate, and isolates from other countries were grouped in a separate subgroup of this group. Also in the phylogenetic tree, the Northeast isolates and the Fars and Kohgiloyeh & Boyer-Ahmad province isolates were classified in Group II.

The results of this research indicate that the virus is endemic and that its likely origin was in Iran and then spread to other parts of the world. The impact of geographic segregation on the evolution of GFLV can also be deduced.

 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.

Pome fruits are affected by many viruses that cause diseases with adverse effects in orchards worldwide. Apple chlorotic leaf spot virus (ACLSV) is one of the most widespread and economically important latent viruses that naturally affect many Prunus species, apples (Malus domestica Borkh.), pears (Pyrus communis L.) and the other rosaceous species. ACLSV infection rates of up to 80–100% in many commercial apple cultivars with yield losses of the order of 30–40% have been reported. In most commercial apple cultivars, the infection generally is latent, but insensitive cultivars, such as apple trees grown on Marubakaido (Malus prunifolia cv. Ringo) rootstocks, malformation and reduction in leaf size and chlorotic rings or line patterns are common. The severity of symptoms induced by ACLSV depends largely on the plant species and virus strains. Some virus isolates induce a severe disease in apricot and plum characterized by depressions and protuberances that deform the fruit, often confused with the “sharka” disease due to Plum pox virus (PPV), and named for this reason as “pseudopox.” ACLSV is a filamentous virus, 680–780 nm long and 12 nm in width as the type species of the genus Trichovirus in the family Betaflexiviridae. ACLSV contains a single-stranded, positive-sense RNA about 7.5–8 kb in size, with a polyadenylated 3" terminus and a cap at its5"-end, and also contain multiple copies of a single coat protein (CP) of 21–24 kDa. The economic importance of ACLSV is largely due to its worldwide distribution and its capacity to induce severe graft incompatibilities in some Prunus combinations, causing major problems in nurseries. In apple trees, ACLSV frequently is detected in coinfection with Apple stem grooving virus and Apple stem pitting virus. ACLSV is mainly transmitted by grafting. No natural virus vectors are known for this virus and are not known to be seed or pollen transmitted. The older National Apple Collection of Native and Imported apple cultivars in Kamalshahr Horticulture Research Station located in Karaj includes 85 cultivars and promising genotypes on seed stocks benefit a high genetic variability. Though the cultivars were screened for more biotic and abiotic factors, but no screening has been achieved yet for virus diseases, while this valuable germplasm comprises a wide range of newly released cultivars or in releasing procedure, high yield natives and those imported selected as adapt to Iranian climate, so a great need of healthy primary nucleus to establish mother orchards for certified plant material. As this valuable germplasm is very important in providing healthy primary nucleus to establish mother orchards for certified plant material so the aim of this research was an evaluation of native and imported apple tree cultivars of this Collection to ACLSV infection.

To assess the occurrence and the prevalence of this virus in the collection, a total of fifty accessions were collected. The sample collection was carried out in summer (2016) and spring (2017). The collecting method consisted of sampling leaves homogeneously distributed around the canopy of the plant. All samples were screened for the presence of ACLSV by DAS-ELISA using the ACLSV specific polyclonal antibody using commercial kits purchased from Bioreba Company, Switzerland. To confirm ELISA results some cultivars were subjected to reverse transcription-polymerase chain reaction. Total RNA was isolated from some apple cultivars using CTAB RNA extraction method and was used as a template for RT-PCR. Specific oligonucleotide primers corresponding to a region of the ACLSV genome that encodes part of the CP, were used in RT-PCR. The amplified PCR products were analyzed in 1% agarose gel stained by ethidium-bromide and visualized under UV light after electrophoresis.

The ELISA results showed that 39 out of 50 cultivars were infected by ACLSV. RT-PCR on total RNA from the ELISA positive samples resulted in the amplification of an expected 358 bp DNA fragment. In RT-PCR out of 18 tested cultivars, five were infected. Some cultivars including Makintash, Red Rome Beauty, Starking, Ardebil1, Nayan Arangeh, Khorsijan, Red spur cooper, Yellow transparent 1, IR6-1 and Ardebil2 recognized ACLSV free in ELISA and RT-PCR, so could be used in the production and employment of virus-free propagating material program after infection testing to the other three important viruses Apple stem pitting virus, Apple stem grooving virus and Tomato ringspot virus. The results showed a high rate of ACLSV infection of native and imported apple cultivars in the older National Apple Collection in Kamalshahr Horticulture Research Station. Previous studies, in apple gardens and nurseries of Iran, have shown a high percentage of ACLSV infection too. Similar results have also been obtained on the percentage of infection with this virus in the Czech Republic, Romania, Albania, and Bosnia and Herzegovina. As germplasm exchanges are the main source of transmission of new viruses to countries, one of the most important strategies to control viruses in fruit trees is to prevent the introduction of virus-infected germplasm into the country.

During the growing seasons of the years 2013-2014, Alborz, Mazandaran, Markazi and Tehran provinces were surveyed to assess the distribution of PNRSV and ToRSV in rose plants, field bindweed and common sow thistle weeds grown nearby the sampled roses. A total numbers of 600 symptomatic and asymptomatic rose leaves and 50 weeds were sampled. Using with the DAS-ELISA serological test, samples were tested. Results indicated that roses are infected to PNRSV with the infection rates of 39.24%, 30.7%, 44.8% and 38.1% and to ToRSV with the infection rates of 38%, 25.6%, 32.3% and 28.3% through in Tehran, Alburz, Mazandaran and Markazi respectively.  It was also revealed that PNRSV and ToRSV was respectively 26% and 18% incidence in weeds. Infected samples were mechanically inoculated on herbaceous propagative and indicator plants using with the potassium phosphate buffer pH 7.1 containing the antioxidant materials. To confirm the presence of viral infections in diseased roses and indicator plants, infected plants were analyzed by RT-PCR, using PNRSV-specific primers to amplify a related portion of the PNRSV and ToRSV genomes. Those infected samples have positively reacted with the both specific primers and desired DNA fragments with the size of about 210bp for the coat protein of PNRSV and 500bp for the polymerase of ToRSV were amplified for them. By this research and for the first time, presence of PNRSV and ToRSV in rose plant and herbaceous weeds was assessed.

Wisteria vein mosaic virus (WVMV) is a member of the Potyvirus genus (Potyviridae) with flexuous filamentous particles. The disease was first reported by Brierley & Lorentz (1957) in the USA from W. floribunda. They cited reports of similar cases dating back to the 1940s and suspected that a virus was responsible for the disease. Afterward the virus has been reported in Wisteria spp. in Australia, New Zealand, China, Germany, Czech Republic, Italy and in the Netherland. In comparison to the majority of potyviruses, WVMV has a very narrow host range, naturally infecting only Wisteria spp. (W. sinensis, W. floribunda, and W. venusta). This virus can be introduced into the landscapes on new plants that were infected during their production. Other wisteria can become infected by mechanical inoculation if infected sap is moved on cutting tools or by aphids during feeding from sap of these plants. In terms of symptoms induction, the virus can cause systemic, local chlorotic (sometimes necrotic) lesions or mottling and mosaic on the leaves of wisteria spp. plants and other experimental hosts of Chenopodium spp., Nicotiana megalosiphon and several legumes. The virus is serologically related to Bean common mosaic virus (BCMV), Bean yellow mosaic virus (BYMV), Clover yellow vein virus (ClYVV) and Watermelon mosaic virus (WMV). WVMV is transmitted by aphids, grafting or mechanical means with no significant impact on ornamental host plant except inducing an unfavorable leaf appearance in the landscape. In August 2017, a plant of Wisteria sinensis in the campus of Ferdowsi University of Mashhad in Iran was observed to have chlorotic, mottling and mosaics on some leaves, which resembled the symptoms caused by wisteria mosaic disease. RNA was extracted from the symptomatic plant with an RNeasy Plant Mini Kit (Qiagen, Germany) and tested by RT-PCR using potyvirus degenerate primers CIF/R. Amplicons of the expected size, 680 bp were obtained. The PCR product was cloned and then sequenced. The partial sequence of Nib gene was also amplified (350 bp) by degenerate primer for potyvirus Nib gene and the obtained fragment were cloned and then sequenced. A BLASTn search in the GenBank showed 90.64% nucleotide sequence identity with WVMV Chinese isolate (AY656816) followed by Watermelon mosaic virus (80.67%) and Soybean mosaic virus (80.46%). The least identity was with an isolate of Lettuce mosaic virus (56.34%). It seems that the Chinese and Iranian isolates of this virus are closer to each other but due to lack of nucleotide sequence from other part of the world in CI and Nib genes it is hard to conclude that Iranian isolate is just close to Chinese isolate and it should be considered that may other isolates exist which are closer to Iranian WVMV isolate than Chinese isolate. From this research, it can be concluded that this virus is present in wisteria spp. plant in Khorasan Razavi province of Iran. In terms of sequence similarity based on partial nt sequence of CI gene (680 nt), it is most similar to a Chinese isolate of WVMV (90.64 %) followed by WMV (80.67 %) and SMV (80.46 %) in nt level and within BCMV subgroup of potyviruses. It seems likely that the disease is spread primarily through vegetative propagation rather than by aphid or mechanical transmission. Although the disease does not markedly reduce the vigour of infected plants, the foliage of such plants is chlorotic and mottled, rendering them unsaleable or can cause the ornamental plants as unfavorable host.

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.

Infections with predominant viruses including Cucumber mosaic virus (CMV), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) and Tobacco mosaic virus (TMV) in tomato fields of Azarbaijan-e-Sharqi Province were surveyed. 205 samples showing symptoms such as mosaic, dwarfing, necrosis, leaf narrowing, leaf and fruit deformation were collected from ….and subjected to glasshouse inoculations. Samples suspected to be infected by CMV were first screened against the virus by ELISA, and then RNA were extracted. RT-PCR reactions were performed by the use of CMV-, Tospovirus or Tobamovirus specific primers depending on virus symptoms. Upon optimization of the annealing temperature in the PCR the anticipated bands were amplified corresponding to Tospovirus, Tobamovirus or CMV in 6, 37 and 23 samples, respectively. Restriction analyses with HindIII, EcoRI and XbaI of the fragments which were amplified with Tospovirus-specific primers showed, TSWV wasin all the six samples. In addition, cloning and sequencing of the amplified fragments proved the TSWV identity of the isolates. When the fragments amplified with the Tobamovirus primers were restricted by TaqI the resultant restriction profile revealed ToMV identitiy of the virus in 37 samples, but no TMV was present in the samples. Also, MspI restriction digestion of the fragments amplified with the CMV primers showed that the CMV isolates were placed in Subgroup I. In addition, the resultant data were indicative of high frequency of infection with CMV and ToMV in the collected samples.

Potato virus Y is one of the most important damaging viruses with a broad host range. In this research due to detection and isolation of Potato virus Y in turnip fields of Khuzestan province during 2014 growing season, characteristics of three selected isolates (7Tu, 9Tu and 10Tu) from Dezful, Shushtar and Shush cities were investigated. To analyze the host range of the isolates, three selected isolates were mechanically inoculated on some members of Solanaceae, Chenopodiaceae, and Amaranthaceae. All three isolates induced local and systemic symptoms on the leaves of potato, tomato, pepper, datura, and gomphrena plants, but no symptoms were observed on Chenopodium quinoa. Analysis of the PVY partially purified preparations indicated that molecular weight of two protein bands obtained by SDS-PAGE was estimated to be about 33 and 31 KDa. To determine the strain of selective isolates the specific primer pairs CF/OR, NF/NR, OF/OR, and NF/OR were used. Using OF/OR and PVY3TIM/PVNIBIP primers, two fragments with 609 and 1200 bp length respectively were amplified. The CP region in the all three isolates was amplified, cloned and sequenced. The results of all aforementioned tests showed that 7Tu, 9Tu, and 10Tu isolates are probably a member of NTN or W strain groups. According to our knowledge, this is the first report on occurrence of PVY in turnip fields.

Cachexia (Citrus cachexia viroid, CCaVd) and exocortis (Citrus exocortis viroid, CEVd) are the most important citrus viroid diseases in the world. Detection of the viroids through the common serological methods is not feasible and the current methods are time consuming and costly with some limitation. Purification and accurate detection of the viroids was studied by column chromatography with CF-11 cellulose powder in presence of ethanol (common method for viruses’ dsRNA detection) in comparison to the conventional methods. During 2010-2012, sampling was done from twigs of 22 Mandarin and 15 orange trees have symptoms of cachexia and exocortis, respectively, in East Mazandaran. Leaf samples were ground and mixed with extraction buffer. Purification carried out in CF-11 column and viroid molecules extracted with ethanol and precipitated by adding sodium acetate. The nucleic acid extracts were analyzed by 1% agarose gel electrophoresis and specific bands of the two viroids with molecular sizes of 7000-7800bp (circular form) and 300-400bp (linear form) were detected in warm and all months, respectively. The dsRNA nature of the bands was confirmed by nuclease treatment (DNaseI and RNaseA) and 2M LiCl extraction methods.Viroid entity of the each nucleic acid sample was recognized by RT-PCR method using specific primers of cachexia and exocortis viroids. 500 orange and 300 mandarin seedlings were tested in healthy citrus seedling production program.As regards of advantages of this method (repeatability, reliability, saving time and price) and confirmation by RT-PCR, the method is recommended for viroid detection in healthy citrus seedling production programs.

Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) are important viruses in tomato fields. In this research, 36 tomato plant samples showing viral symptoms were collected from fields in various regions of west and northwest of Iran. To detect CMV and TSWV, cDNAs were prepared using Random Hexamer primer and total RNAs extracted from the collected samples. The prepared cDNAs and specific primers for CMV-coat protein (CP) gene and TSWV-nucleocapsid (N) gene were used to amplify a part of each virus genome by Polymerase Chain Reaction (PCR), separately. The results revealed that a 654 bp fragment from the CMV-CP and a 777 bp fragment from TSWV-N were amplified from six and three samples, respectively. To detect mixed infection of CMV and TSWV in tomato plants simultaneously, we used their specific primers in a single PCR assay. DNA fragments from both viruses were amplified from two samples. In these plants more severe symptoms such as deformation, mosaic, chlorosis and necrosis on the leaves were observed and compared to the specific symptoms in plants infected by either CMV or TSWV. Phylogenetic tree of mixed isolates based on nucleic acid and amino acids showed that the isolate of CMV in this research were grouped with isolates from Iran and India whereas TSWV isolate were grouped with isolates from different country such as Turkey, Italy and Montenegro.

Tomato (Lycopersicum esculentum Mill.) is one of the most important crops in Iran and throughout the world. In the previous survey conducted in 2013, PVY was introduced as the dominant virus in Yazd (Iran). In the present study, the partial genome sequence of a selected isolate called T5 was determined and analyzed. Following total RNA extraction of the T5 isolates, three fragments with 824, 1700 and 837 bp lengths were amplified using CPr/CPf, M4T/Sprimer and P1/P2 primer pairs respectively in RT-PCR. According to the nucleotide and amino acid sequences comparison of the CP, NIb and P1 regions of the isolate with the other PVY isolates in the Genebank, T5 is a member of NTN strain.