Soybean mosaic virus (SMV) which belongs to the virus family Potyviridae, causes a disease in soybean that is present in soybean-growing areas of the world, and is widely distributed in northern Iran. Detection of SMV is very important for disease management. In the present study several serological and molecular (nucleic acid- based) methods of rapid virus detection were compared. Serological studies including DASELISA, DAC-ELISA, TPIA and DIBA were optimized and compared to identify the virus by using a polyclonal antibody. Among the serological methods, TPIA and DIBA are simple and TPIA is rapidly and easily applicable in the field. However, TPIA was found to be preferable. TPIA is time-saving, not requiring conventional sap extraction and also nitrocellulose membranes used for printing can be used in the field and stored for a long time or transported to other laboratory to be processed. RT-PCR and Immunocapture RT-PCR (IC-RT-PCR) were performed as molecular methods for detecting SMV using a pair of primers designed to amplify a fragment in the coding region of the SMV coat protein. To extract total RNA for RT-PCR, two methods including RNAWIZ and phenolchloroform were used. A part of the coat protein genome of SMV was converted to cDNA using a reverse transcription (RT) reaction. For IC-RT-PCR method, virus partial purification was carried out by solid-phase (0.2 ml microfuge tube) adsorbed polyclonal antibody, and then the RT reaction was carried out in the tube. In both methods cDNAs were amplified by PCR. Both methods amplified the expected fragment in virus-infected plants. Whereas RT-PCR requires total RNA extraction, ICRT- PCR does not have total RNA extraction problems. Our findings suggest that TPIA and IC- RT- PCR can be routinely used for SMV detection, with high efficiency.

The reaction of sixteen potato-promising clones to PVX, PVA and PVY was evaluated in greenhouse condition. At first, viral infection of each clone was examined using DASELISA. Infected clones were treated by electrotherapy technique to achieve virus free clones. Single nodes of healthy plants were cultured on MS medium. After growing, plantlets were transferred to greenhouse and minitubers harvested about two months later were used as experimental materials. Three week old potato plants were inoculated mechanically with tobacco plants extracts infected to PVX, PVY and PVA. After 30 days, the result of DAS-ELISA of each plant was scored. Data analysis showed that the reaction of clones to the three viruses were significantly different at 1% level. However, the difference among plantlets in each replicate was insignificant. Mean comparison of clones, ELISA absorbance values test indicated that clones 397015-1 and 397015-13 were resistant to all the three viruses and clone 397008-2 was susceptible to three viruses. Other clones were resistant or susceptible to one or two viruses.

The full-length coat protein gene of Grapevine fanleaf virus (GFLV) isolates from Iran was characterized by reverse transcription polymerase chain reaction (RTPCR) and sequencing. The expected 1515 bp coat protein (CP) gene amplicon was obtained for 16 isolates out of 89 that were identified by double antibody sandwich enzyme-linked immune sorbent assay (DASELISA) in a population of 330 symptomatic grapevine leaf samples. CP products of eight isolates were cloned and nucleotide sequences were determined.Parsimonious trees indicated that GFLV isolates from Iran formed a distinct cluster, suggesting an independent evolution.

Potato virus Y (PVY), Potato virus X (PVX), Potato virus S (PVS) and Potato leaf roll virus (PLRV), are the most important viruses of potato and can cause serious diseases and significantly reduce the yield and quality of potato crops. The incidence of these viruses is reported from almost all main potato growing regions in Iran. Therefore, application of a sensitive, rapid and inexpensive procedure for simultaneous detection of potato viruses is very important for management of their control. In the present research, a sensitive and reliable multiplex RT-PCR technique for the detection of PVY, PVX, PVS, PLRV and 18S rRNA as internal control was employed and compared with ELISA, the method used routinely to assay potatoes for virus infections. Total RNA was extracted from virus infected potato leaves. Then cDNA for above viruses and 18S rRNA was individually synthesized using their specific reverse primers and single RT-PCR for each virus and 18S rRNA. Subsequently, multiplex RT-PCR was carried out with five primer pairs in a single reaction. As a result, five different fragments (145-342 bp) specific to the viruses and the internal control were simultaneously amplified and were identified on the basis of their molecular sizes. The sensitivity of our optimized system also was compared with that of commercial DAS-ELISA for detection of PVY, PVS and PLRV. The results showed that the sensitivity of multiplex RT-PCR was 100-fold greater for detection of PVY, PVS and PLRV and the duplex RT-PCR was 1000-fold greater for detection of PVY than that of commercial DASELISA.

In order to investigate the Iris yellow spot virus (IYSV) Sampling from onion fields and greenhouse of Khorasan Razavi province in summer of 2009 was done. Totally 435 samples were collected from this onion fields and 142 from ornamental plants (Rose, Gladiol, Iris, Plargonium, Chrysanthemum, Begonia, Petonia and Carnation). The plants that had symptoms such as chloratic, necrotic and diamond shape lesions were collected and transferred under cold condition to labratory then tested by DAS-ELISA and the sap of posetive plants inoculated to 4 coultivar of indicator plants that cultivated in greenhouse Nicotiana rustica (leef deforming and systhemic chloratic and necrosis),N. benthamiana, N. clevelandii, N.tabacum var Samson (systhemic chloratic and necrosis) then the inoculated indicator plants tested by DAS-ELISA, sap of positive indicator plants inoculated to 4 cultivars of onion include in yellow of Neishabour, white of Neishabour, red of Dargaz, red of dorche of Isfahan. The symptoms similar to the symptoms on infected onion in the fields appeared in their plants. For molecular detection, RNA extraction was done by PEG6000 Precipitation and RNX ™(plus ) kit.In RTPCR tests, specific primers designed for nocleoprotein gene amplified 181 bp and 139 bp fragments.. DASELISA test results have indicated that all of the onion fields were infected with the virus in various rates. IYSV has been detected in 107 samples of onion, 7 samples of chrysanthemum floweres and 1 sample of iris floweres. This is the first repot of Iris yellow spot virus on onion and chrysanthemum in Iran.

Golestan province is one of the main tobacco growing regions in the country. Tobacco Streak Virus (TSV) is a destructive pathogen on tobacco that has a wide host range and occurs worldwide. In order to serological detection of TSV at 13 region of Golestan province, 500 infected samples belong to 8 families; Chenopodiacae, Amaranthaceae, Fabaceae, Solanaceae, Malvaceae, Poaceae, Cucurbitaceae and Asteraceae with leaf distortion, stunting, mosaic, yellowing, necrosis and terminal bud blight symptoms, were collected and tested by the DASELISA method using specific polyclonal antibody. The results showed 78 positive reactions out of 500 samples in ELISA tests. The infection of peanut, dahlia, tobacco, mung bean, common bean, soybean, pepper and potato was positive to TSV, and negative for other plants in ELISA test. Some of ELISA positive samples were selected and their extracts in phosphate buffer 0.1M, pH 7.4 containing mercaptoethanol was mechanically inoculated on indicator plants, tobacco, fat-hen and tomato. TSV infection was confirmed by ELISA on indicator plants. The mechanical inoculation of different isolates of 8 mentioned hosts on tobacco and tomato, caused similar symptoms.

Pokeweed (Phytolacca Americana L.) is a plant native to American countries which is widely used across the continent. Despite the probability of having digestive toxicity, this plant has an important role in many therapeutic applications for humans and plants. This plant has a capacity to control some plant and human pathogens due to the presence of ribosome-inactivating proteins (RIPs) in this plant. In this work, the antiviral effect of aqueous pokeweed leaf and stem aqueous extracts against Potato virus Y, one of the most important viruses infecting potatoes, was studied in tobacco model plants. The presence of the pokeweed antiviral protein (PAP) in the extract was confirmed by SDS-PAGE analysis. Results of serological assay (DASELISA) showed lower absorbance values for the pokeweed treated samples as compared with non-treated, PVY-infected control plants, indicating lower levels of PVY infections in the treated plants. Results showed that the aqueous extract of pokeweed, at concentration of 0.14 ppm, had an inhibitory effect against PVY infection in tobacco model plants with a maximum controlling effect up to four days post-inoculation.

Pierce's disease (PD) has been recently reported from Iran. The aim of this study was to detect this disease in major vineyards in Iran. To determine the distribution pattern of this disease, during 2013-2018 years, 365 samples of leaf and stem of grapes of 88 vineyards were collected with PD symptoms. Samples obtained of Iranian grape growing provinces of Fars, Khorasan Razavi, North Khorasan, South Khorasan, Semnan, Alborz, Tehran, Qazvin, Zanjan, West Azarbaijan, East Azarbaijan, Kurdistan, Kermanshah, Lorestan, Kohgiluyeh va BoyerAhmad, Markazi, Hamedan and Chahar Mahal va Bakhtiary. Samples were tested for the presence of Xylella fastidiosa by culturing, DAS-ELISA and PCR assay with X. fastidiosa-specific primers. Pathogenicity tests, for some isolates, were conducted on grapevine (cv. Bidaneh Qazvin) under greenhouse conditions. Early symptoms is leaf scorched, while adjacent tissues turned yellow or red. Some grapes showed “ matchstick” symptoms in which the leaves dropped from the plant, while petioles remained attached. Approximately 56% (212 of 365), 44. 8% (43 of 96) and 7. 1% (26 of 365) of the samples, by DASELISA, PCR and culturing, were infected to X. fastidiosa, respectively. Although based on DAS-ELISA all of the sampled provinces showed infected to X. fastidiosa, there were by the results of culturing and PCR infected to X. fastidiosa is definitive for some vineyards provinces of Fars, Khorasan Razavi, Alborz, Qazvin, Zanjan, Lorestan, Hamedan and Chahar Mahal-va-Bakhtiary.

Persian lilac (Melia azedarach,Family: Meliaceae) and fig (Ficus carica,Family Moraceae) are important medicinal plants native to Iran. In this study, the antiviral effect of the crude ethanolic and aquatic extracts of the persian lilac and fig plants in decreasing disease severity caused by Cucumber mosaic virus (CMV) in cucumber plants was assessed using with the double antibody sandwich ELISA (DASELISA) and semi quantitative-RT-PCR (Sq-RT-PCR). In addition, the transcription level of PR-1 gene and specific activity of phenylalanine ammonia-lyase (PAL) enzyme were evaluated using Sq-RT-PCR assay. ELISA results showed that the presence level of CMV in cucumber seedlings was significantly decreased up to 15 days after their treating with the 100 ppm of ethanolic and 1000 ppm of aquatic extracts of fig and persian lilac plants in compare to the non-treated control plants. The PR-1 expression level and PAL enzyme specific activity were also significantly increased compared to the non-treated control infected plants in samples treated with the ethanolic extracts of persian lilac and fig plants at the concentration of 100 ppm up to 15 days after treatments. Disease severity was also significantly decreased in cucumber seedlings treated with the 100 ppm concentration of ethanolic extract of the persian lilac and fig plants. Totally, results of this study indicated that the systemic acquired resistance might be activated against CMV by applying ethanolic extracts of persian lilac and fig plant used in this study in treated cucumber seedlings through different molecular and biochemical mechanisms.