Comparative transmission of two cucumber mosaic virus isolates by two color morphs of Acyrthosiphon pisum (Harris).

UNLABELLED:  Cucumber mosaic virus (CMV) is one of the most important legume-infecting viruses, which is transmitted effectively by pea aphid Acyrthosiphon pisum (Harris) (Hem: Aphididae). Transmission efficiency of two CMV isolates (As and Kh from cowpea and bean hosts, resp.) by red and green color morphs of pea aphid were evaluated on bean plants. Triple-antibody sandwich ELISA (TAS-ELISA) using CMV-specific monoclonal antibodies revealed that both CMV isolates belonged to the serotype II. Bean plants inoculated by viruliferous aphids were assayed by double-antibody sandwich ELISA (DAS-ELISA) at 16 days post inoculation (dpi). The results showed that the transmission rate of CMV-As by the red morph was significantly higher than by the green morph, resulting in significantly higher transmission rate of the virus (As + Kh) by the red morph than by the green morph, with p≤ 0.1. Similarly, the efficiency of CMV transmission by the red morph of A. pisum was higher than the efficiency of transmission by the green morph. The higher transmission rate and efficiency of CMV by red pea aphid would be important in the epidemiology. Based on these results, we hypothesize that the transmission efficiency of CMV is affected more by the difference in transmission determinants of A. pisum color morphs than by the sequence of virus coat protein determinants. KEYWORDS: Aphididae; Bromoviridae; color polymorphism; transmission efficiency.

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.

Identification of Three Strains of Bean common mosaic necrosis virus in Common Bean from Iran.

Bean common mosaic virus (BCMV; genus Potyvirus) has been recognized as a major constraint on bean production in Iran. BCMV and Bean common mosaic necrosis virus (BCMNV) have been reported from bean-growing regions of Iran (2,3), but no attempts were made to differentiate strains of BCMNV. During the early growing seasons of 2003 and 2004, 141 bean leaf samples suspected of BCMV infection were collected from the main bean-producing regions in Tehran Province (Varamin, Damavand, Boein Zahra, Hashtgerd, and Karaj). Symptoms included mild and severe mosaic, leaf curling, malformation, vein-banding, vein-clearing, mottle, and blister on the leaves. In addition, seeds of green bean and Chiti bean (300 each) were obtained from seed lots in Tabriz (East Azarbaijan) and Miyando-Âb (West Azarbaijan) and planted in the greenhouse. Emerging seedlings were subsequently screened for BCMV infection by ELISA and sodium dodecyl sulfate-Ouchterlony double diffusion test. Anti-BCMV polyclonal antisera used in this study included those raised specifically against NL-1, NL-3, NL-4, NL-6, NL-5, NL-8, and NY-15 strains. Seedborne viral infection on newly emerged seedlings varied (2 to 5%) depending on the province and bean cultivar. Seedborne symptoms were characterized as leaf curling, malformation, and necrosis. Among indicator plants used for host range determination, symptom characterization, and biological purification of BCMV, only Chenopodium quinoa, C. amaranticolor, and Phaseolus vulgaris L. cv. Red Kidney developed chlorotic local lesions in response to the BCMV inoculation. Further, P. vulgaris L. cvs. Bountiful, Red Kidney, and Stringless Green Refugee developed leaf mosaic and malformation as a systemic reaction to the inoculation. Of 172 isolates of BCMV investigated, seven representative strains, designated as A (37.2%), B (11%), C (9.3%), D (7.5%), E (12.2%), M (7.5%), and N (15.1%), were selected on the basis of symptom development on the indicator plants and serological tests with strain-specific polyclonal antisera. Thermal inactivation point, dilution end point, and longevity in vitro of the selected BCMNV strains were in the range of 60 to 65°C, -3-(-4), and 3 to 4 days, respectively. Pathogenicity groups of the selected strains were determined by symptom response (sensitive or resistance) at 26 and 32°C in the bean differential host range (1). The designated strains B and E from Tehran Province were assigned to standard strain NL-3 or pathotype VIa, strains A, C, and D from Tehran Province were assigned to standard strain NL-5 or pathotype VIb, and strains M and N from Azarbaijan Province were assigned to standard strain NL-8 or pathotype III. Western immunoblot analysis of viral capsid protein revealed that unlike NL-8, the BCMNV strains NL-3 and NL-5 had the apparent molecular mass of 32 kDa, which was slightly less than that of reference strain NL-8 (33 kDa), thus further confirming that these strains belong to serotype A of BCMV (e.g., BCMNV). Electron microscopy study showed that the virion particles were flexuous, filamentous, and unenveloped. To our knowledge, this is the first report of the differentiation of BCMNV strains from Iran. References: (1) E. Drijfhout. Page 1 in: Agriculture Research Report 872. Centre for Agriculture 46 Publishing and Documentation. Wageningen, the Netherlands, 1978. (2) W. J. Kaiser and G. H. Mossahebi. Phytopathology 64:1209, 1974. (3) N. Shahraeen et al. Plant Dis. 89:1012, 2005.

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.

Characterisation of lettuce virus X, a new potexvirus infecting lettuce in Iran.

A virus with flexuous rod-shaped particle morphology was found in samples from lettuce during a survey of viruses infecting lettuce in Tehran province in Iran. This virus was subjected to a complete analysis of its biological and molecular features. The entire nucleotide sequence of the virus was determined, revealing a polyadenylated ssRNA genome consisting of 7,212 nucleotides [without poly (A) tail] and possessing an organization typical for potexviruses. Comparative genome analysis showed that the lettuce virus is closely related to Alstroemeria virus X, narcissus mosaic virus and asparagus virus 3. Based on particle morphology, physico-chemical properties and the complete genome sequence, this virus is a member of a new species in the genus Potexvirus, for which the name lettuce virus X (LeVX) is proposed. Biological assays using an infectious cDNA clone and a wild-type isolate of LeVX revealed that the virus, despite reaching high concentrations in all lettuce cultivars tested, does not cause symptoms in lettuce.

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.

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.

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.

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 and distribution of lettuce mosaic disease in Tehran province from Iran.

Lettuce mosaic virus (LMV) Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) were identified in fields of Tehran province. In this study 452 leaf samples were collected from the fields throughout the Tehran province during 2002 and 2003. Distribution of Lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV)and Arabis mosaic virus (ArMV) was determined with DAS-ELISA. Percentage of single Infection to LMV. CMV or TSWV was 20.58, 15.93 and 9.96% respectively. Also 15.28% of samples were co- infected with LMV+CMV, 8.19% with LMV+TSMV and 7.74% with CMV+TSWV. 4.65% of samples were Infected to all of these three viruses. LMV was found in 48.69%, CMV in 43.59% and TSWV in 30.54% of samples totally. Therefore LMV is major dominant agent of lettuce mosaic disease in Tehran province. This is the first report of occurrence of TSWV on lettuce in Iran and first report of CMV and LMV in Tehran province.

Biological and molecular characterization of lettuce mosaic virus from Tehran province in Iran.

In this study, lettuce samples having LMV infection symptoms were collected from Tehran fields during 2003. Samples tested for LMV infection by immuno printing. Three positive samples in immuno printing collected and their characteristics were determined. In mechanical inoculation, these Isolates produced symptoms on Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Nicotiana benthamiana, Lactuca sativa cv. Mantilia and cv. Terocadero (which contains the mol1 resistance gene and susceptible respectively), but not cv. Salinas 88 (which contains the mol2 resistance gene). LMV was purified and LMV polyclonal antiserum was produced in rabbit by a series of intravenous and intramuscular injections, the precipitin titre of this antiserum was 1:1024. Gel double diffusion test (GDDT) was performed, and precipitin bands appeared. SDS-PAGE and western blotting showed the presence of coat protein 29 kDa. In IC-RT-PCR with on LMV specific primer pair, an approximately 1300 bp fragment was amplified.