Characterization of a filamentous virus from Bermuda grass and its molecular, serological and biological comparison with Spartina mottle virus.

Bermuda grass with mosaic symptoms have been found in many parts of Iran. No serological correlation was observed between two isolates of this filamentous virus and any of the members of the family Potyviridae that were tested. Aphid transmission was demonstrated at low efficiency for isolates of this virus, whereas no transmission through seed was observed. A DNA fragment corresponding to the 3' end of the viral genome of these two isolates from Iran and one isolate from Italy was amplified and sequenced. A BLAST search showed that these isolates are more closely related to Spartina mottle virus (SpMV) than to any other virus in the family Potyviridae. Specific serological assays confirmed the phylogenetic analysis. Sequence and phylogenetic analysis suggested that these isolates could be considered as divergent strains of SpMV in the proposed genus Sparmovirus.

Carnation mottle virus, an important viral agent infecting carnation cut-flower crops in Mahallat of Iran.

One of the most important cut-flower crops grown worldwide on commercial scale is Carnation (Dianthus caryophyllus L.). It's the main production of Mahallat where is one of the most important ornamental plants production centers of Iran. Infection of carnation with pathogens Like viral agents causes economic losses in carnation cut-flower crop. One of the viral agents of this flower is Carnation mottle virus (CarMV) which is the type member of genus Carmovirus and belongs to the Tombusviridae family. It is naturally transmitted by grafting and contacting between plants. Although its infection lead to mild symptims, it weakens the plant to infection by other pathogens. The carnation greenhouses of Mahallat were visited during 2008 January to April and 100 samples with mild mosaic symptom were collected and tested by DAS-ELISA using CarMV specific polyclonal antibody. The results showed that 75% of samples wrere infected with this virus. Mechanical inocubation of Chenopodium quinoa, C. amaranticolor and Spinacea oleracea with extracted crude sap of CarMV infected carnation Leaves in phosphate buffer (pH, 7) resulted in appearance of chlorotic and necrotic local lesions on inoculated leaves 4-7 days after incubation. The virus was partially purified using C. amaranticolor locally symptomatic leaves. Total soluble proteins were extracted from healthy and CarMV infected C. amaranticolor plants and beside partially purified preparation electrophoresed through 15% poly acrylamide get according to SDS-PAGE standard procedure. Protein bands were electroblotted onto nitrocelluse membrane and incubated with CarMV polyclonal during western immunoblot analysis according to standard method. The result revealed a distinc protein band with Mr of 35.5 kDa in total protein preparation of infected plant and viral partial pure preparation, without any reaction in those of healthy plant. RT-PCR carried out using total RNA extracted from infected plant by Rneasy Plant Mini Kit (Qiagen)and a pair of primers, CPu, CPd, corresponding to the flanking region of the virus CP resulted in amplification of a DNA fragment in expected size around 1 kbp.

Virus diseases in the tobacco fields of Guilan and Western Azerbaijan provinces of Iran.

Tobacco (Nicotiana tabacum L.) is one of the important industrial plants in Iran. Viruses as an important group of plant pathogens cause many losses on the quality and quantity of tobacco crop. There was few information on the types of plant viruses infecting the tobacco fields of Guilan and almost no information for Western Azerbaijan province. During 2005-2007, leaf samples were taken from symptomatic plants in the growing areas of these two provinces. The observed symptoms on plants in the fields varied from mild mosaics to severe necrosis. The regions of sampling were including Rasht, Bazar-jomeh, Soumae-Sara, Talesh and Astara in Guilan and Ourmia, Sardasht and Ghara-Ziaeddin in Western Azerbaijan. The tobacco types and varieties from which the samples were taken included air-cured burley variety Burley 21 and to a lesser extent, oriental tobacco variety Basma Serres in W. Azerbaijan and flue-cured varieties Coker 347 and Virginia El in Guilan province. Samples were tested by DAS-ELISA method (Clark and Adams, 1977) using the polyclonal antibodies for a set of tobacco viruses. Some samples with positive reactions in DAS-ELISA were inoculated to indicator test plants such as Chenopodium amaranticolor, Datura metel, D. stramonium, Physalis floridana, Nicotiana rustica, N. glutinosa, and tobacco (varieties White burley and Samsun). The results of greenhouse experiments were consistent with serological tests. The following viruses which are listed in order of their overall abundance within the tested samples were detected: Tobacco streak virus (TSV), Tomato spotted wilt virus (TSWV), Tobacco etch virus (TEV), Tobacco ringspot virus (TRSV), Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). In all samples more than one virus infection was detected. The most severe mosaic type symptoms including the deformation and blistering on leaves were mainly seen in the infections by CMV and TMV. The most severe necrotic type symptoms including necrosis of midribs or veins and in some cases stem necrosis were generally associated with the infections by PVY and TSWV. Except TMV infection which was not detected in the Burley 21 variety in W. Azerbaijan, the above mentioned viruses were present in all sampling regions. The lack of TMV infection on Burley 21 is due to the presence of N gene, conferring resistance in this variety.

High incidence of tobacco streak virus in the tobacco fields of Iran.

Currently the northern provinces of Iran (Mazandaran, Golestan and Gilan) are the main tobacco growing regions in the country and this crop has an special importance in the national economy. Three tobacco types including flue-cured (mainly Coker 347 and some Virginia E1), burley (Burley 21) and oriental (Basma 178-2) are presently grown in these regions. Epidemics of viral diseases have occurred during the recent years in many tobacco fields in these areas. The quality of tobacco products which is much important, is adversely affected by plant pathogens specially viruses. In a survey on the viruses of tobacco, the fields in these regions were inspected and leaf samples from symptomatic plants were collected. Some plants had one or more of the symptoms such as dentate leaf margin, thicker leaf tissue and necrotic areas on the stem. The samples were tested for TSV infection by the DAS-ELISA method (Clark and Adams, 1977) using polyclonal antibody (AS-0615, DSMZ, Germany). TSV was detected in more than 79% of all tobacco samples from these three provinces. The TSV infection level among the tested samples was 86.8% in Gilan (Rasht, Bazar-Jomeh and Talesh), 82.3% in Mazandaran (Behshahr, Sari, Neka and Sourak) and 71.8% in Golestan (Gorgan, Aliabad and Minoodasht). No significant difference was seen among the infection levels for the mentioned commercial varieties and also some other tested varieties such as C176, K326 and MN944. It seems that there is no resistance sources against this virus within these varieties. Also the results of tests for TSV were similar in two consecutive years (2004 and 2005). It should be added that not all of the TSV infected plants showed the stated symptom types. Many of the TSV infected samples had mixed infections with one or more other viruses such as TSWV, CMV, PVY and TMV and there was almost no sample with a single TSV infection. This is the first report on the occurrence and distribution of TSV in the tobacco fields of Iran, too.

Evaluating the efficiency of a TPIA method for the detection of two tobacco viruses.

Field experiments are usually necessary for analyzing the efficiency of control methods, the resistance of varieties and many other virological studies. Such experiments generally include a large number of samples to be tested serologically. DAS-ELISA is a very accurate technique that has been widely used for identifying viral infections. For large numbers of plant samples, it takes a long time for the sample preparation, plate washing and other procedures. In this study, the efficiency of a simple and laborsaving TPIA (Tissue Print Immunoassay) method was evaluated for the identification of two important aphid-transmitted viruses (CMV and PVY) of tobacco fields in comparison with DAS-ELISA as the standard method. The leaf samples were collected from the fields of three commercial tobacco types (flue-cured, burley and oriental). Each sample was divided to two parts and each part was examined by one of the methods. DAS-ELISA was done based on the method described by Clark and Adams (1977). In TPIA, small parts of the leaf samples were rolled and then cut by a sterile sharp blade. The cut surface was gently printed on 1 cm2 blocks drawn on a nitrocellulose paper. Air dried paper was located first in 1% BSA for blocking the empty sites on paper, then in the buffer containing AP-conjugated polyclonal antibody for 3 h and finally in NBT-BCIP solution for color development. Between these stages, the paper was washed thoroughly (three times) by shaking in fresh washing buffer. The results of each sample were recorded and compared with those of DAS-ELISA. By considering DAS-ELISA as the reference method, the sensitivity of TPIA for the detection of PVY and CMV was 96.1% and 92.7%, respectively. The positive results by TPIA which were not detected positive by DAS-ELISA were regarded as false positive. These were 8 (out of 316 tested samples) for CMV and 6 (out of 204 samples) for PVY. Although the results of TPIA were not completely consistent with DAS-ELISA, it seems that this method can be used for some general studies. The most important advantages of this method were that it didn't need sample extraction and was done using only one antibody which was the conjugated antibody of each virus. This method gives more rapid results (within a day) in comparison with DAS-ELISA that needs more time.

Detection of Alfalfa mosaic virus (AMV) in pea field in Iran.

During the spring and summer, in 2003-2004, pea viruses were identified in twenty pea fields of Tehran. Some leaf samples were collected randomly from pea fields of Tehran. Samples were tested by Double Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA) technique using polyclonal antiserum of Alfalfa mosaic virus (AMV), AS-0001, DSMZ, Braunschweig, Germany). The samples were extracted in 0.1 M Phosphate buffer pH 7 to 7.5 and inoculated on Chenopodium amaranticolor, Chenopodium quina, Phaseolus valgaris, Vicia faba, Vignia unguiculata. Pea cultivars were infected by AMV, causing mild mosaic, translucent veins and a diffuse green-yellow of tender parts and spots may also was involved necrosis of tissue. Infected plants grow slowly and malformed pods produce fewer ovules. In Chenopodium amranticolor, C. quina chlorotic and necrotic flecks, and Vicia faba systemic mosaic had produced. Phaselous vulgaris and Viginia unguiculata are good assay hosts for strains that produce local lesions after 3-5 days in these plants. Back inoculated on Pisum sativum and Vicia faba and tested with DAS-ELISA that had been confirmed the results. This is the first report of AMV on pea from Iran.

First report of occurrence of two viruses on pea field in Iran.

An intensive survey was conducted to identify virus diseases affecting pea crops in Tehran province of Iran. A total of 270 pea samples were collected randomly from pea fields. samples were tested by Double Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA) using polyclonal antisera prepared against PSBMV (AS-0129, DSMZ, Braunschweig, Germany) and TSWV (AS-0580, DSMZ, Braunschweig, Germany). Virus disease incidence in pea samples was followed by PSBMV (33%) TSWV (24.4%) and PSBMV+TSWV (17.77). The positive samples with PSBMV were extracted in 0.05M phosphate buffer pH 6.5-7 containing 2% pvp and inoculated on Pisum sativum, Vicia faba, Chenopodium quinoa, Chenopodium amaranticolor. That produced in Pisum sativum; leaflets roll downwards, shoots curl, internodes shorten and plants are rosetted. Early infections reduce flower and fruit formation or eliminate their development. Broad bean has symptoms accompanied by a certain margin rolling and leaflet distortion. In Chenopodium amaranticolor necrotic local lesions and Chenopodium quinoa chlorotic local lesions had produced. The positive samples with TSWV were extracted in 0.01 M phosphate buffer containing 1% Na2 SO3 and inoculated on Petunia hybrida, Pisum sativum. TSWV causes several symptoms in infected peas, including brown leaf petiole and stem coloration, leaflet spotting, vein necrosis. In petunia hybrida after approximately 5 days showed local necrotic lesion. Biological purification in TSWV with chlorotic local lesions in Petunia hybrida and in PSBMV; chlorotic local lesions in Chenopodium quinoa were done. In PSBMV, back inoculated on Pisum sativum and Vicia faba also tested with DAS-ELISA. RT-PCR confirmed the results. This is the first report of PSBMV and TSWV naturally infecting pea in Iran.

Identification of PVY-N on potato and tobacco in Tehran and Mazandaran provinces.

During two growing seasons in years of 2003 and 2004 potato and tobacco of virus infected plants were collected from fields in Tehran (Damavand) and Mazandaran (Behshahr) provinces. Serological methods of TAS-ELISA and DIBA were performed by using PVY antiserum (DSMZ - Plant Virus Collection; Germany) but only PVY was detected. The strain of samples was determined by using MAb of potato virus Y (AS-0403/1; DSMZ; Germany). The molecular weight of the virus coat protein was approximately 34 kDa in SDS-PAGE and Western blotting. Total RNA was extracted for RT-PCR. Immunocapture RT-PCR and RT-PCR products were 974 bp by using specific primers of PVY. IC-RT-PCR has given the best results.

Occurrence and physico-serological properties of potato virus S (PVS) in Iran.

1818 collected samples of potato plant showing virus infection symptoms from 85 fields were tested for PVS infection using DAS-ELISA. Average of infection to this virus varied from 0 to 100%. Least infection was belonging to fields with new introduced varieties. On the other hand native and old introduced cultivars showed heavy infection. In field condition, PVS infected plants didn't show very obvious symptoms, so some infected plants may be missed in field sample collecting. The physical properties of 3 isolates, Avaj, Stanboly and Agria No 15 were determined. TIP 55-60 degrees C, DEP 10(-3) and Liv measured 3-4 days. Ouchterlony agar double diffusion test using SDS was useful for virus detection and precipitation lines didn't show any spur between isolates, although isolates differs slightly in symptomatology. SDS-Page and Western blotting methods used successfully for virus detection and determining and measuring viral protein components.

Purification of potato virus X and preparation of its antiserum.

Potato virus X (PVX) isolated from the potato leaf and tuber samples which were collected from various fields in Damavand and Ardabil. The initial isolations of the virus were made from potato by mechanical inoculation on Gomphrena globosa L. and Chenopodium spp. that produce local lesion, and then it causes mosaic on Nicotiana spp. and Datura stramonium L. An isolate of the virus inoculated to Nicotiana glutinosa L. and it was maintained throughout the work. Sap from infected N. glutinosa was ineffective after dilution to 10-6, 10 minutes at 70 degrees and 10 weeks at room temperature. The virus was readily purified from infected leaves and the best protocol was Moreira & Jones 1980 than the other 2 methods of Fribourg 1975 and Shepard & Shalla 1972. Antisera were prepared against native, degraded proteins and micro precipitin test showed that both antisera had a 1/512 titer. Precipitin lines with D - Protein antiserum was better of the native protein antiserum in agar double diffusion test than treated with SDS. The isolate of the virus was not transmitted by none of 2 species of Cuscuta but transmitted from infected leaves to healthy plants with sap inoculation without using Carburandum. This isolate showed positive reaction with gamaglubulin in kate received from CIP centre.

Study on potato virus M (PVM) occurrence in potato fields in Iran.

57 native potato tuber samples collected from different potato growing region of Iran, planted on single rows in Karaj College experimental station. Plant samples of each single row plus 9.25 Fresh foliage samples collected from fields under new introduced cultivars were tested for potato virus (PVM) infection during growing season. Also 78 weeds and field crops belonging to Solonacae and Leguminosae from or neighboring to potato field were tested. Results indicated that PVM was not found on any plant other than potatoes. PVM was detected on 16 samples of 57 old vars, Virus was not seen in any samples collected from fields under new varieties. Results show that PVM is limiting in this crop. PVM detecting is difficult using assay hosts. Best test plants were French bean var Red kidney, Showing pinpoint necrotic LL, also Datura metel and Nicotiana debneyi are useful for virus detection showing chlorotic local lesion. Also microprecipition and gel diffusion test can be used for virus detection but Elisa was the best method. PVM infected plant showed 11-19.5 percent yield decrease in 3 cultivars tested.

Occurrence, distribution and relative incidence of mosaic viruses infecting field--grown squash in Tehran province, Iran.

Squash (Cucurbita pepo) belongs to Cucurbitaceae family. Every year Cucurbitaceae are planted world wide. They are one of the most important economic crops. Cucurbitaceae are threatened by viruses. Many viruses damage the plants of this family. Since nine viruses have been reported on squash from Iran. In this survey, during 2002--2003, to determine the distribution of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV), 466 samples were collected from squash field in Tehran province. Infected plants showing symptoms such as: mosaic, yellowing, deformation, shoestring of leaves and fruit deformation and yield reduction. Distribution of CMV, ZYMV and WMV were determined by DAS-ELISA. Thepercentage of ZYMV, WMV and CMV were 35.6, 26.1 and 25.1% respectively. Triple infection (CMV+ZYMV+WMV) were found in 6.4% of samples. ZYMV were found the most frequently the viruses. This is the first report of WMV on squash in Tehran province.