Occurrence and distribution of Potato virus Y strain N (PVY(N)) on celery in Iran.

Celery (Apium graveolens) is an important crop grown in many countries. Different types of diseases present a major constraint to Celery production and can lead to significant reductions in yield Potato Virus Y, the type member of the genus Potyvirus(family Potyviridae) is one of the causal agents of viral diseases in crops. In the present study, PVY occurring in celery fields was determined as the causal agent of mosaic disease of celery (Apium graveolens). The virus is naturally transmitted by aphids in a non-persistent manner. During growing seasons 2006-2007 celery fields were visited through Tehran Province and a total 332 sample based on selection of plants expressing symptoms like mosaic, vein clearing and mottling were collected. By using serological method (DAS-ELIZA) with specific antiserum of PVY (DSMZ-AS-0137.403) 17.64% of the samples were infected with PVY .The reaction of PVY infected samples were positive in TAS-ELIZA with specific monoclonal antibody (MAbs) of PVYN strain (DSMZ-AS-403.1). PVY N strain isolated from celery was reported for the first time in Iran and in the world in this survey.

Nodule infection by bean yellow mosaic virus in Vicia faba and molecular characterization of it.

Root nodules infection of different faba bean (Vicia faba L.) cultivars by bean yellow mosaic virus (BYMV) and the effect of the disease on the specific activity of the nodule are reported. The disease reduced the fresh weights of tops, roots, root nodules and induced premature nodule decay and/or nodule drop. Six local cultivars: Barekat, Iranshahri, Saraziri, Aljazayeri, Shakhbozi and Zohre of faba bean were selected and inoculated with BYMV under greenhouse conditions. ELISA test (DAS-ELISA) with specific BYMV antibody (DSMZ AS0471) demonstrated that nodules from faba bean plants which had been inoculated with BYMV contain the virus too. Susceptibility of different faba bean cultivars was analyzed by ELISA. The relative accumulations of BYMV in the nodules were evaluated by mean ELISA values (OD405) of BYMV. There was significantly difference in cultivars. Cultivars went more susceptible from Barekat to Iranshahri, Saraziri, Aljazayeri, Shakhbozi and Zohre. High susceptibility of Zohre was confirmed in a second experiment including visual evaluation and DAS ELISA. Analysis by IC-RT-PCR revealed the presence of the virus in all nodules and amplified a 970 bp fragment with specific designed primers (Forward primer (5'-CT(AC) CA(AG) ATG GAG AA(CT) CC(CT) GC 3') and Reverse primer (5'-CCA AAG TTC CAA TCA CCA CC 3').

Determination of seed-born percentages of bean common mosaic necrosis virus (BCMNV) in three genotypes of Phaseolus vulgaris.

Bean common mosaic necrosis virus (BCMNV) is one of the most damaging viruses of bean that naturally transmitted by aphid in non persistent manner and through the seeds. BCMNV belongs to the genus Potyvirus and the family Potyviridae. During the growing season of 2004, bean leaf samples with viral symptoms were collected from Tehran province, Karaj region. DAS-ELISA by using BCMNV polyclonal antiserum (AS-0239, prepared in DSMZ, Germany) was conducted and samples with viral infection were distinguished. IC-RT-PCR was done to amplify the cp gene of isolates. The nucleotide sequence of one isolate was determined and analysis of this and other published sequences confirmed this isolate as BCMNV. The confirmed isolate was inoculated on three bean genotypes (butter bean ks-21478, kidney bean ks-31170, navy bean ks-41235) using 0.01 M Potassium phosphate buffer (pH = 7). After appearance of symptoms, the inoculated plants were tested by DAS-ELISA and IC-RT-PCR. In DAS-ELISA test, 68% infection of butter bean and kidney bean genotypes and only 7% infection of navy bean genotype were confirmed. In IC-RT-PCR by using specific primers (NL3), a 922 bp fragment was amplified in all genotypes, even symptomless plants and the ones which were negative in ELISA test. To determine the percentages of infected seed, harvested seeds were planted. Most of the seedlings in two-leaf stage died with black root symptoms. All seedlings were tested by DAS-ELISA and IC-RT-PCR. The results of these assays showed that the percentage of seed infected was 78%.

Occurrence of viruses infecting pea in Iran.

A survey was conducted to determine the incidence of Alfalfa mosaic virus (AMV), Bean yellow mosaic virus (BYMV), Broad bean wilt virus-1 (BBWV), Pea leafroll virus (PLRV), Pea enation mosaic virus (PEMV), Pea seed borne mosaic virus (PSbMV), Potato virus x(PVX), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) on pea (Pisum sativum) in Iran. A Total of 1276 random and 684 symptomatic pea samples were collected during the spring and summer of 2002-2004 in Tehran province of Iran, where pea is grown, and tested by enzyme-linked immunosorbent assay (ELISA) using specific polyclonal antibodies. Serological diagnoses were confirmed by electron microscopy and host range studies. Incidence of viruses in decreasing order was PVX (69%), ToMV (59%), PSbMV (36.6%), BBWV-1 (26.1%), BYMV (20.3%), AMV (17.77%), TSWV (12.6%), PEMV (10.9%), PLRV (6.78%). In this survey, natural occurrence of AMV, BBWV-1, PSbMV, TSWV, PVX and ToMV was reported for the first time on the pea in Iran.

New indicator plants for potato virus X.

For over 2 years (2002-2004); a sever virus like disease on leaves in Pisum sativum was observed from Tehran in Iran. The identity of which was established by the following host reactions and serological and molecular assays. Collected samples were tested for the presence of the virus using the DAS-ELISA (direct double antibody sandwich-ELISA). Leaf sap of each test samples was diluted 1:10 in ELISA sample buffer. The samples reacted in ELISA with antibody against Potato virus (x)(PVX). The samples were passed through three single chlorotic local lesion transfers on Gompherena globosa, which showed chlorosis 5 to 7 days after mechanical inoculation. Finally one local was homogenized in phosphate buffer (0.05 M, pH 7.2) and rubbing the inoculums on carbourundum--dusted leaves of Nicotiana glutinosa. For host range studies extracts prepared from infected N. glutinosa leave were inoculated to the 35 species. The virus induces systemic mosaic symptoms on Petunia hybrida, Physalis floridana, Nicandera physaloides and Solanum nigrum. This has not been yet record as assay hosts for PVX. Total RNA extracted from symptomatic, plants and RNA extracted from purified virus preparations were tested using reverse transcription polymerase chain reaction (RT-PCR) with specific primers designed to amplify a fragment of the RNA-dependent RNA-polymerase gen. Back inoculation from different symptomatic plants, had been done to Pisum sativum and confirmed by DAS-ELISA. This is the first report of Petunia hybrida, Physalis floridana, Nicandera physaloides and Solanum nigrum as new indicator plants for PVX.

Comparison of biological and molecular characterization of Iranian lettuce mosaic virus isolates.

Lettuce mosaic virus (LMV) is one of the most damaging viruses in lettuce and endive cultivating regions. In order to review the characteristics of different LMV isolates of Iran during 2004-2005 samples were collected from lettuce fields in Esfahan, Ghom, Khorasan, Khuzestan and Tehran provinces. All of the isolates were detected by LMV polyclonal antiserum (AS-0155, DSMZ Germany) in ELISA and TIPA tests. Biological purification was done for the LMV isolates and then they were maintained and propagated on Chenopodium quinoa. A range of plant species such as C. amaranticolor, C. album, Carthamus tinctorius, Gazania sp., Gomphrena globosa, Pisum sativum, Spinacia oleracea were inoculated with these isolates using potassium phosphate buffer (0/05M). Molecular weight of coat protein was determined by Polyacrylamid gel electrophoresis (PAGE). Immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) was performed using LMV polyclonal antiserum and specific primer pairs of LMV as described by Zerbini et al. (1995). The amplified fragments were included the whole CP and 3'UTR regions and the nucleotide sequences of them determined. All isolates induced chlorotic local lesions on C. amaranticolor and chlorotic local lesions with symptoms of systemic infection (vein clearing) on C. album. Tehran isolate in addition, caused local lesions on Gomphrena globosa with red border and white centre. This isolate infected Pisum sativum without any symptoms. Back inoculation on C. quinoa and DAS-ELISA confirmed the latent infection. None of these isolates infected Carthamus tinctorius, Gazania sp. and Spinacia oleracea. The molecular weight of coat protein was determined 30.33 kDa. Western-blot proved this band as the coat protein of the virus. IC-RT-PCR amplification of LMV isolates produced the expected size IC-RT-PCR product of 1300 bps. The comparison of nucleotide sequences showed that there were 98% identities.

Identification of prevalent potyvirus on maize and johnsongrass in corn fields of Tehran province of Iran and a study on some of its properties.

During a growing season in 2004, 231 leaf samples of virus infected and mosaic and dwarf mosaic symptoms showing maize (Zea mays L.) plants and 258 leaf samples of mosaic showing johnsongrass (Sorghum halepens L.) plants from various corn fields in Tehran province were collected. Serological tests of DAS-ELISA and DIBA were performed on samples using antisera of sugarcane mosaic virus (SCMV), maize dwarf mosaic virus (MDMV), sorghum mosaic virus (SrMV) and johnsongrasss mosaic virus (JGMV). In both tests performed on leaf samples extractions, all samples reacted strongly with SCMV antiserum and no reaction was seen with other 3 potyviruses antisera. 0.1 M potassium phosphate buffer (pH = 7) containing 2% polyvinyl pyrrolidon (PVP) was used for mechanical inoculation and all isolates were inoculated and propagated on sweet corn cv. Pars 403 and grain sorghum cv. Kimia. In serological tests on the inoculated plants samples also only SCMV was detected. Purification of virus was done using a modified "minipurification" method and the concentration of purified virus was 11.45 mg/ml and ratio of A260/280 = 1.2 was calculated for it. Electron microscopic study using ISEM and decoration method with SCMV antiserum revealed filamentous flexuous particles of SCMV. In SDS-polyachrylamide gel electrophoresis and Western blot test using SCMV antiserum that were performed on infected samples and purified viruses, the molecular weight of the virus coat protein was approximately 37-38 KDa and a difference among the CP weights of various SCMV isolates was not found. Reverse transcription-polymerase chain reaction (RT-PCR) was done using SCMV F3 and SCMV R3 primers and amplified fragments of approximately 900 bp in size were as in expected. The host range study with selected isolates of SCMV showed that the virus isolates were not transmitted by mechanical inoculation on Avena sativa, Panicum miliaceum, Setaria italica, Pennisetum americanum, Hordeum vulgare and Triticum aestivum. The isolates produced red-brown necrotic streaks on sudangrass (Sorghum sudanense) that lately changed in systemic necrosis. In host reaction studies on sorghum (Sorghum bicolor) cultivars, the virus isolates caused severe necrotic and killer reaction on sorghum cultivars Payam, Sepideh and Speed feed, but caused systemic mosaic and non-killer reaction on sorghum cultivars Kimia, KFS2, KFS3 and Jumbo. The present study showed that SCMV is the prevalent potyvirus and the main causal agent of mosaic and dwarf mosaic on maize plants in province. Since the virus is prevalent on johnsongrass plants in marginal areas of corn fields too, it seems that the origin of the virus on corn is from johnsongrass and the virus is a special strain of sugarcane mosaic virus that infects johnsongrass too.

Potato virus yisolated from pepper fields in Tehran Province.

Potato Virus Y was known as the main cause of yellowing and vein necrosis of pepper in Tehran Province, using Double Antibody Sandwich Elisa (DAS-ELISA). Biological properties including host range of the isolate was determined after biological purification. Host range studies showed that pepper isolate of PVY caused vein clearing and mosaic symptoms on Datura metel and Capsicum annum, mosaic on Nicotiana tabacum cv. White Barley, N. tabacum cv. Samsun and N. rustica but didn't show any symptoms on Physalis floridana, Chenopodium amaranticolor, C. quinoa and Solanum tuberosum. Also the virus was physically purified from propagative hosts: Datura metel, Nicotiana tabacum cv. White Barley and Capsicum annum using Leiser & Richter (1978) method. The A260/280 absorbance ratio of the isolate was 1.16, 1.50 and 1.04 for purified preparations from D. metel, N. tabacum cv. White Barley and C. annum respectively. SDS-PAGE of the coat protein extracted from purified virus preparations gave bands at position of about 34 KD and Western Blotting (using PVY antiserum with 1/1000 dilution, obtained from DSMZ, Germany) confirmed its as the PVY coat protein. In order to prepare antiserum, five injections were given at 7-10 days intervals to rabbit. A week after the last injection the rabbit was bled and the antiserum collected. The primer pairs NIA/F and NIA/R (Glais et al., 2005) were used in IC-RT-PCR and the length of the amplified fragment was 752 bp. This is the first report of PVY incidence in pepper fields in Iran.