Cucumber Mosaic Virus Infection Research Articles

Kajian Plant Growth Promoting Rhizobacteria sebagai Agens Proteksi Cucumber Mosaic Virus dan Chilli Veinal Mottle Virus pada Cabai

This study was conducted to evaluate the effectiveness of plant growth promoting rhizobacteria (PGPR) in protecting chillipepper plant from infection of cucumber mosaic virus (CMV) and chilli veinal mottle virus (ChiVMV). Seven isolates of PGPR, i.e. BC1, BTP2H, BTP3G, BTP3O BTP1, BTP2D, and T1F were applied as seed treatment and soil drench. Plants height, number of branch, and fruits weight were measured every one and ten weeks after virus inoculation. Virus concentration in plants and disease incidence were confirmed by enzyme-linked immunosorbant assay (ELISA). Results showed that inoculation with PGPR improved the seed germination. Eight days after sowing, the percentage of PGPR treated seed germination reached 50-84%; whereas those of untreated seed reached only 18%. In general, PGPR treatment significantly reduced (p ≤ 0.05) the effect of virus infection on plant growth. Two PGPR isolates, i.e. BTP1 and BTP2H, maintained fruit weight of infected plants as good as those of healthy plants. Based on ELISA, PGPR was able to inhibit the disease incidence. The BTP3O and BTP2D isolates even protected the plant from ChiVMV infection. Concentration of salicylic acid and peroxidase were relatively higher on plants treated with PGPR than those without PGPR treatment. This gave an indication that PGPR may act as induction agents for systemic acquired resistance. Therefore, PGPR treatment is a promising strategy to control viral diseases on chillipepper.

Effects of Virus Infection ofMimulus guttatus(Phrymaceae) on Host Plant Quality for Meadow Spittlebugs,Philaenus spumarius(Hemiptera: Cercopidae)

Herbivorous insects often feed on pathogen-infected plants in nature, and it is likely that pathogen infection alters host plant quality. We documented the effects of plant infection by a widespread plant virus on host plant quality for a generalist insect herbivore and tested the hypothesis that these effects vary among plant genotypes. We found that infection by Cucumber mosaic virus (CMV) altered the host plant quality of Mimulus guttatus (Phrymaceae) for meadow spittlebugs, Philaenus spumarius (Hemiptera: Cercopidae). The effects of CMV infection on host plant quality, however, varied among full-sib M. guttatus families, suggesting that these effects vary among plant genotypes. In most full-sib families, CMV infection either had no effect on host plant quality or increased host plant quality as measured by spittlebug size and development time. In a few M. guttatus families, however, CMV infection decreased host plant quality. There was no relationship between the effect of CMV infection on plant growth and the subsequent effect of CMV infection on spittlebug performance, suggesting that broad changes in host plant performance (e.g., growth rate) were not responsible for the effects of CMV infection on host plant quality for spittlebugs. We suggest that future studies of the effects of pathogen infection of plants on insect herbivores consider variation among plant genotypes in the mechanisms and ecological consequences associated with these effects.

Three Previously Unrecorded Viral Diseases of Astilbe, Fuschia, and Thermopsis Species in Minnesota

Interest in virus diseases of perennial ornamentals has been increasing because of their increasing monetary value, because wholesale producers perceive an advantage in marketing disease-free stock, and because widespread international movement of these plants carries the risk of introduction of exotic viruses. In an ongoing study to identify and document viral diseases of perennial ornamentals used in the United States commercial horticultural industry, three virus-like diseases of astilbe (Astilbe chinensis), fuschia (Fuschia cv. Gartenmeister) and false lupine (Thermopsis caroliniana) occurring in Minnesota were investigated. Symptomatic plants were selected from lots in commercial greenhouses and garden centers in several locations in Minnesota. Astilbe with systemic chlorosis was found to be infected with Tobacco ringspot virus (TRSV). Fuschia with leaf mottling and leaf deformation was found to be infected with Cucumber mosaic virus (CMV), and false lupine with mosaic and leaf deformation symptoms was found to be infected with Bean yellow mosaic virus (BYMV). Identification of the three viruses was based on: 1) virion presence and morphology in partially purified leaf extracts using electron microscopy (EM) and immunosorbent electron microscopy (1); 2) enzyme-linked immunosorbent assay using crude leaf extracts; and 3) biological properties, including symptoms produced in indicator plants. Antisera to BYMV (ATCC PVAS-368), CMV (ATCC PVAS-30), and TRSV (ATCC PVAS-157) were obtained from the American Type Culture Collection, Manassas, VA. No other virus-like particles were observed with EM in partially purified leaf extracts of the three plants, no virus-like particles were observed in similar preparations from asymptomatic plants, and indicator plant tests did not indicate the presence of any other mechanically transmissible viruses. The TRSV isolate from astilbe and the BYMV isolate from false lupine produced typical symptoms on indicator plants susceptible to known isolates of these two viruses. The CMV isolate from fuschia was similar to previously described isolates of CMV (2) in most respects and was readily transmitted in a nonpersistent manner by Myzus persicae, but was unusual in that it did not infect Nicotiania benthamiana, N. glutinosa, and tomato, which are normally highly susceptible to infection by CMV. The identity of the fuschia CMV isolate was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR) amplification with CMV-specific oligonucleotide primers (3). The PCR product was of the predicted size (500 bp) and was cleaved by restriction digestion with EcoRI, suggesting that the fuschia virus is a Type II CMV isolate (3). To my knowledge, this is the first report of TRSV infection in astilbe, CMV infection in fuschia, and of a viral disease of false lupine.

Analysis of natural populations and possible natural reassortment of Cucumber mosaic virus

Plants naturally infected with Cucumber mosaic virus (CMV) were collected and analyzed by electrophoresis of the replicative form of dsRNA and by Northern blot hybridization using CMV RNA-specific probes. Some of the CMV-infected plants, especially winter crops, contained two kinds of RNA 1 segments or RNA 2 segments (or both), suggesting that mixed infections of CMV occurred naturally. Single-aphid-transmitted isolates (SATIs) from the field isolate containing two RNA 1 segments were grouped into three types by the electrophoretic mobility of RNA 1 (i.e., those containing one slow segment, those containing one fast segment, and those containing both). Furthermore, SATIs and single-lesion isolates, generated from the plants inoculated with a mixture of two CMV isolates that could be differentiated by their electrophoretic dsRNA profiles, were analyzed by dsRNA, indicating that nonparental progenies were observed. These results suggested that genetic reassortment of CMV RNA may occur in nature and that this is an important mechanism in CMV evolution.

Occurrence ofCucumber mosaic virus on vanilla (Vanilla planifolia Andrews) in India

Cucumber mosaic virus (CMV) causing mosaic, leaf distortion and stunting of vanilla (Vanilla planifolia Andrews) in India was characterized on the basis of biological and coat protein (CP) nucleotide sequence properties. In mechanical inoculation tests, the virus was found to infect members of Chenopodiaceae, Cucurbitaceae, Fabaceae and Solanaceae. Nicotiana benthamiana was found to be a suitable host for the propagation of CMV. The virus was purified from inoculated N. benthamiana plants and negatively stained purified preparations contained isometric particles of about 28 nm in diameter. The molecular weight of the viral coat protein subunits was found to be 25.0 kDa. Polyclonal antiserum was produced in New Zealand white rabbit, immunoglobulin G (IgG) was purified and conjugated with alkaline phosphatase enzyme. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was standardized for the detection of CMV infection in vanilla plants. CP gene of the virus was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR), cloned and sequenced. Sequenced region contained a single open reading frame of 657 nucleotides potentially coding for 218 amino acids. Sequence analyses with other CMV isolates revealed the greatest identity with black pepper isolate of CMV (99%) and the phylogram clearly showed that CMV infecting vanilla belongs to subgroup IB. This is the first report of occurrence of CMV on V. planifolia from India.

Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library

Immunomodulation by the ectopic expression of intracellular antibodies ('intrabodies') has a great potential for interfering with physiological or pathological functions in vivo in a highly specific manner. One of the major obstacles of this technology is the inability of most antibodies to properly fold and function in the reducing environment of the cytoplasm, which prevents the formation of essential disulfide bonds. We wished to assess the intracellular performance of antibodies derived from a semi-synthetic single-chain variable fragment (scFv) phage display library ('F8 library') built on a thermodynamically stable single-framework scaffold. To this purpose, we chose to modulate the infection of a pandemic plant pathogen, the cucumber mosaic virus (CMV). After in vitro 'biopanning' on immobilized virions, two scFvs were biochemically characterized, showing high affinity toward the antigen. They were transiently expressed at high yields as soluble molecules in the cytoplasm of Nicotiana benthamiana plants. Subsequently, they were expressed in the cytoplasm of transgenic tomato plants. Challenge with high viral dose showed that both scFvs were able to elicit a phenotypic effect and led to the identification of a transgenic line fully resistant to infection. In these plants, the scFv binds the virus in the inoculated leaves preventing viral long distance movement. This work represents the first demonstration that the 'F8 library' can be directly screened in vitro to rapidly isolate antigen-specific scFvs that act as effective intrabodies in vivo. These antibodies represent powerful tools to interfere with several intracellular targets, modulating pathogen infectivity and/or cellular metabolism.

Allamanda Mosaic Caused by Cucumber mosaic virus in Taiwan.

Allamanda (Allamanda cathartica L., family Apocynaceae) is native to Brazil and is a popular perennial shrub or vine ornamental in Taiwan. Plants showing severe mosaic, rugosity, and leaf distortion symptoms on leaves are common in commercial nurseries and private gardens. Examination of crude sap prepared from symptomatic leaves using an electron microscope revealed the presence of spherical virus particles with a diameter of approximately 28 nm. The virus was mechanically transmitted to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). The virus caused local lesions on inoculated leaves of Chenopodium quinoa and C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, Nicotiana benthamiana, N. glutinosa, N. rustica, and N. tabacum. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Tests of leaf sap extracted from naturally infected allamanda and inoculated indicator plants using enzyme-linked immunosorbent assay were positive to rabbit antiserum prepared to CMV. Viral coat protein on transblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis reacted with CMV subgroup I specific monoclonal antibodies (2). With primers specific to the 3'-half of RNA 3 (1), amplicons of an expected size (1,115 bp) were obtained in reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from infected allamanda and N. benthamiana. The amplified fragment (EMBL Accession No. AJ871492) was cloned and sequenced. It encompasses the 3' part of the intergenic region of RNA 3 (158 nt), CP ORF (657 nt), and 3' NTR (300 nt) showing 91.8-98.9% and 71.4-72.8% identities to those of CMV in subgroups I and II, respectively. Results of MspI-digested restriction fragment length polymorphism patterns of the RT-PCR fragment and the nucleotide sequence analysis indicate that the CMV isolate from allamanda belongs to subgroup IB, which is predominant on the island. To our knowledge, CMV is the only reported virus that infects allamanda and was first detected in Brazil (3), and this is the first report of CMV infection in allamanda plants occurring in Taiwan.

Identification of the cis-acting elements in Arabidopsis thaliana NHL10 promoter responsible for leaf senescence, the hypersensitive response against Cucumber mosaic virus infection, and spermine treatment

There are 45 NDR1/ HIN1-like ( NHL) genes in Arabidopsis thaliana. Of these, we have focused on the NHL10 gene because of its unique expression profiles: its transcripts are accumulated in a senescence-associated manner, during the hypersensitive response (HR) triggered with an avirulent Cucumber mosaic virus (CMV) strain, and by treatment with the polyamine spermine (Spm [M.S. Zheng, H. Takahashi, A. Miyazaki, H. Hamamoto, J. Shah, I. Yamaguchi, T. Kusano, Up-regulation of Arabidopsis thaliana NHL10 in the hypersensitive response to Cucumber mosaic virus infection and in senescing leaves is controlled by signalling pathways that differ in salicylate involvement, Planta 218 (2004) 740–750]). Here we have performed an analysis of NHL10 promoter using the β-glucuronidase ( GUS) reporter gene-fusion constructs in transgenic A. thaliana plants. With the histochemical staining assay, it was demonstrated that the region between −697 and +53 from the putative transcriptional start site of the NHL10 promoter was essential for responses to leaf senescence, HR to CMV and Spm treatment. These data were confirmed by northern analysis using the GUS reporter gene as a probe. By further deletion analysis, all the cis-acting elements responsive to leaf senescence, CMV-induced HR, and Spm treatment, were narrowed down to the identical −198 to −93 region. It also demonstrated that the controlled expression of NHL10 gene at a transcriptional level.

Population Dynamics of Cucumber mosaic virus in Melon Crops and in Weeds in Central Spain

ABSTRACT Understanding epidemiology and evolution of plant viruses requires knowledge of their ecology: obtaining information about the dynamics and genetic structure of viral populations in their various hosts is necessary to analyze host specialization and to identify reservoirs and inoculum sources. We present here a 3-year analysis of the population dynamics of Cucumber mosaic virus (CMV) in Central Spain in weed hosts and melon. CMV infection in weed hosts was detected throughout the year and showed a clear seasonality, with maximum incidence of 20 to 30% in summer and autumn. The dynamics of CMV incidence were different in various weed habitats (i.e., fallow fields, edges, and wastelands), which differed in stability due to the degree of human intervention. CMV incidence in weed habitats was correlated with the amount of vegetation, estimated both as biomass and as soil cover by plants. CMV population dynamics in melon crops was unrelated to that in weed habitats. Genetic characterization by ribonuclease protection assays and biological characterization on differential hosts showed no significant differences in the frequency of genotypes or biotypes for CMV isolates from weeds or melon. Hence, different population dynamics in various hosts and habitats has not resulted in population differentiation, suggesting that migration between hosts and habitats prevents fragmentation of the population.

Spatial analysis for exclusive interactions between subgroups I and II of Cucumber mosaic virus in cowpea

The dynamics of virus interference in Cucumber mosaic virus (CMV) infection in cowpea were investigated by tissue-blotting and in situ hybridization. Using co-inoculation assays, we discovered that spatial competition between CMV-LE (subgroup I) and CMV-m2 (subgroup II) occurred in the inoculated leaves. Interestingly, competitive interactions between the two viruses also could be observed in the non-inoculated upper leaf tissues of the plants. Furthermore, the pattern of exclusive distribution was observed between challenge and protecting viruses in the serially inoculated leaves. Taken together, it is suggested that the dynamics of competitive interactions between the two subgroups could be characterized by exclusive infection and multiplication of the individual viruses in cowpea plants.

Forecasting aphid outbreaks and epidemics of Cucumber mosaic virus in lupin crops in a Mediterranean-type environment

Cucumber mosaic virus (CMV) causes a serious disease of narrow-leafed lupin ( Lupinus angustifolius). It is seed-borne in lupin and seed-infected plants act as the primary virus source for secondary spread by aphid vectors within crops. Infection with CMV causes yield losses of up to 60% in epidemic years, but has little impact on yield in years when spread is limited. Aphids also cause sporadic yield losses due to direct feeding damage. A simulation model was developed to forecast aphid outbreaks and epidemics of CMV in lupin crops growing in the ‘grainbelt’ of south–west Australia, which has a Mediterranean-type climate. The model uses rainfall during summer and early autumn to calculate an index of aphid build-up on weeds, crop volunteers and self-regenerating annual pastures in each ‘grainbelt’ locality before the growing season commences in late autumn. The index is used to forecast the timing of aphid immigration into crops. The subsequent aphid build-up and movement within the crop, spread of CMV from virus-infected source plants within the crop, yield losses and percentage of harvested seed-infected are then calculated. The model evaluates the effects of different sowing dates, percentages of CMV infection in seed sown and plant population densities on virus spread. The model simulations were validated with 14 years’ field data from six different sites in the ‘grainbelt’, representing a wide range of pre-growing season rainfall scenarios, sowing dates, percentages of infection in seed sown and plant population densities. The model was incorporated into a decision support system (DSS) for use by lupin farmers and agricultural consultants in planning CMV management and targeting sprays against aphids to prevent direct feeding damage. The inputs required from the user are lupin cultivar, anticipated emergence date, percentage CMV infection in seed sown, plant density and location. The output consists of a personalised risk forecast for the user and includes predictions for date of first aphid arrival, aphid numbers, CMV spread, final virus incidence, yield loss due to infection and percentage infection in harvested seed. Predictions from the DSS are accessible via an Internet site and from other information sources. The model can serve as a template for modelling similar virus/aphid vector pathosystems in other regions of the world, especially those with Mediterranean-type climates.

Up-regulation of Arabidopsis thaliana NHL10 in the hypersensitive response to Cucumber mosaic virus infection and in senescing leaves is controlled by signalling pathways that differ in salicylate involvement.

The Arabidopsis thaliana (L.) Heyhn. genome contains 45 genes that show substantial sequence similarity to the tobacco ( Nicotiana tabacum L.) HIN1 (harpin-induced) gene and the Arabidopsis NDR1 (non-race-specific disease resistance) gene. Of these, the nine ( NHL1 to NHL3, NHL8 to NHL12 and NHL22) that bear the highest identity to HIN1 were selected and their expression in various situations was examined. We found that NHL10 behaves like the tobacco HIN1 gene in that its transcripts are abundant in senescing leaves and they specifically accumulate during the hypersensitive response (HR) caused by exposure to an avirulent Cucumber mosaic virus (CMV) strain. Spermine, a novel inducer of tobacco PR (pathogenesis-related) genes, was found to up-regulate expression of NHL3, NHL10 and NDR1. Green fluorescent protein-fusion experiments indicated that NHL2 and NHL10, and possibly NDR1 are localized in the chloroplasts. Studies using Arabidopsis mutants defective in salicylic acid (SA), ethylene and jasmonic acid signalling revealed that the senescence-associated expression of NHL10 is mediated by a pathway that involves SA but that NHL10 expression during CMV-induced HR and spermine treatment is totally independent of SA.

Involvement of RNA2 for systemic infection of Cucumber mosaic virus isolated from lily on zucchini squash

A lily strain of Cucumber mosaic virus (LK-CMV) was not able to systemically infect zucchini squash ( Cucurbita pepo), while Fny strain of CMV (Fny-CMV) caused systemic mosaic and stunting symptom at 4 days post-inoculation on the same host species. The pathogenicity of LK-CMV in zucchini squash was investigated by reassortments of genomic RNAs of LK-CMV and Fny-CMV for infection, as well as by pseudorecombinants generated from biologically active transcripts of cDNA clones of LK-CMV and Fny-CMV, respectively. The assessments of pathogenicity for LK-CMV indicated that RNA2 of LK-CMV was responsible for systemic infection in zucchini squash. In the protoplast of zucchini squash, the RNA accumulations of all constructed pseudorecombinants were indistinguishable and LK-CMV replication was slightly lower than that of Fny-CMV, suggesting that the inability of LK-CMV to infect squash plants was responsible for the poor efficiency of virus movement, rather than the reduction of replication function.

Cucumber mosaic virus Diagnosed in Desert Rose in Florida.

Desert rose (Adenium obesum (Forssk) Roem. & Schult.) is a member of the family Apocynaceae and characterized by fleshy leaves and stems and colorful flowers. This popular, exotic ornamental, originally from southeastern Africa, is propagated vegetatively and is a perennial in warm climates. Virus-like foliar symptoms, including a mosaic with dark green islands surrounding the veins and chlorosis on the leaf margins, were observed on desert rose samples from two southwest Florida nurseries in November 2002. Cucumber mosaic virus (CMV) was identified in symptomatic plants by serological testing for the presence of CMV coat protein with a commercially available ImmunoStrip test (Agdia, Elkhart, IN). A third sample expressing similar symptoms was observed in southeastern Florida in February 2003. The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich enzyme-linked immunosorbent assay (Agdia). An agent was mechanically transmitted from the third sample to Chenopodium quinoa, resulting in the formation of chlorotic local lesions. Examination of inoculated C. quinoa leaves by double-stranded (ds) RNA analysis and electron microscopy (leaf dips) revealed the presence of a typical cucumovirus dsRNA profile and spherical virions ~28 nm in diameter, respectively, providing additional confirmation of a CMV infection. A possible satellite RNA of ~350 nucleotides was also observed by dsRNA analysis. To our knowledge, this represents the first report of CMV infection of desert rose.

In spite of induced multiple defense responses, tomato plants infected with Cucumber mosaic virus and D satellite RNA succumb to systemic necrosis.

Cucumber mosaic virus (CMV) D satellite RNA (satRNA) attenuates the symptoms induced by CMV in most plants, but causes leaf epinasty and systemic necrosis in tomato plants, where programmed cell death (PCD) is involved. However, our understanding of the cellular and molecular responses to the infection of CMV D satRNA that result in this lethal disease remains limited. In this article, we show for the first time, by histochemical and molecular analysis, that multiple defense responses are specifically induced in CMV and D satRNA (CMV/D satRNA)-infected tomato plants but not in mock-inoculated or CMV-infected plants. These responses include callose deposition and hydrogen peroxide accumulation in infected plants. Furthermore, the transcription of several tomato defense-related genes (e.g., PR-1a1, PR-1b1, PR-2, and PR-10) were activated, and the expression of tomato PR-5 and some abiotic and biotic stress-responsive genes (e.g., catalase II and tomato analogs of Arabidopsis AtBI-1 and tobacco hsr203j) are enhanced. The activation and increase in expression of these genes is correlated with the appearance of leaf epinasty and the development of systemic necrosis in infected tomato plants, while increased expression of the hsr203j analog precedes the development of any disease symptoms. The spatial and temporal expression patterns of these genes as detected by RNA in situ hybridization point to the involvement of a complex developmental program that accompanies disease development resulting from CMV/D satRNA infection.

The susceptibility and response of inbred and outbred Mimulus guttatus to infection by Cucumber mosaic virus

The deleterious effects of inbreeding have been well documented, but only recently have studies begun to explore the consequences of inbreeding for important ecological interactions. We examined the effects of inbreeding on the interaction between host and pathogen using the mixed-mating Mimulus guttatus (Scrophulariaceae) and Cucumber mosaic virus (CMV). Inbred (self) and outbred M. guttatus from two California populations (M5 and M13) were rub-inoculated with CMV and compared to sham-inoculated controls. Flower production by outbred plants in host population M5 showed little effect of the inoculation treatment, but inoculation reduced flower production of inbred plants by 12%, indicating that inbreeding reduces tolerance to CMV infection. This interaction fell short of significance, however. The effects of inbreeding and CMV inoculation on biomass in M5 varied significantly across the 15 families used in this experiment, indicating genetic variation in the effect of inbreeding on resistance or tolerance to CMV. CMV infection reduced biomass in host population M13, but there were no significant interactions between virus treatment and level of inbreeding for either flower production or biomass. Enzyme linked immunosorbent assay (ELISA) was used to detect CMV in host tissues. In both populations, mean ELISA absorbance values of inoculated plants were nearly identical for self and outcross hosts, indicating equal susceptibility to CMV. In outbred plants of population M5, flower production did not change with increasing ELISA absorbance, but in inbred plants it declined, indicating reduced tolerance to CMV infection. The results from this study suggest that pathogens may become increasingly detrimental as host populations become more inbred.

Development of CMV-and TMV-resistant chili pepper: field perfermance and biosafety assessment

Both cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) coat protein (CP) genes have been transferred to chilli pepper (Capsicum annuum var. Longunt) cultivar 8212 by a modified procedure of Agrobacterium tumefaciens-mediated transformation using hypocotyl as the explant. PCR analysis revealed the presence of both CMV and TMV CP genes in at least 11 primary transformants out of 49 kanamycin-resistant chili pepper plants. Ten T1 lines from five independent transformation events were identified as putative homozygous transgenic lines based on the rooting assay of their T2 seedlings on the kanamycin-containing media. Integration and expression of CMV CP and TMV CP transgenes in one of the homozygous line, 16-13, were confirmed bySouthern blot, RT-PCR and western blot analyses. Line 16-13 was highly resistant to infection of homologous CMV and TMV strains in greenhouse conditions when successively challenged with CMV and TMV or challenged with TMV alone.Futhermore, field trials on T2, T3 and T4 progenies of Line 16-13 were performed on scales of 123, 300 and 10,000 plants, respectively, in consecutive years 1996, 1997 and 1998 with the permission of the Chinese government authority. The transgenic plants displayed delayed symptom development and significantly milder disease severity in field conditions when compared to untransformed chili pepper plants, resulting in 47 and 110% increase in pepper fruit yield in surveys conducted in 1997 and 1998 trials, respectively. Finally, quality analysis and biosafety assesment were performed on transgenic chili pepper fruit concurrently with the control fruit, and demonstrated that the transgenic chili pepper fruit is substantially equivalent to the non-transgenic pepper in terms of the quality and biosafety when consumed as a food additive.

First report of the natural occurrence of eight viruses affecting soybeans in Iran

During the summer of 1999/2000 a survey of viruses infecting soybeans was conducted in five main soybean cultivation provinces of Iran (Golestan, Mazandaran, Ardebil, Lorestan and Khuzestan). A total of 3110 leaf samples were collected randomly from 80 soybean fields (30–50 samples per field). Samples were tested by the enzyme-linked immunosorbent assay (ELISA) for the presence of 13 viruses using antisera against Alfalfa mosaic virus (AlMV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Soybean mosaic virus (SMV), Tobacco mosaic virus (TMV), Tobacco ringspot virus (TRSV) and Tomato ringspot virus (ToRSV) (Sanofi Pytho-Diagnostics, 92430 Marnes-la-Coquette, France); Bean common mosaic virus (BCMV) (Adgen Ltd, Auchincruive, KA6 5HW, UK); Tobacco streak virus (TSV) (As-0171, PV-0309) and Tomato spotted wilt virus (TSWV) (As-0526, As-0580, PV-0526) (DSMZ, BBA Messeweg 11/12, D-38104 Braunschweig, Germany); Pea enation mosaic virus-1 (PEMV-1) (Iranian pea isolate provided by S. Farzadfar); Blackeye cowpea mosaic virus (BlCMV) (from Dr Pinnow, Department of Plant Pathology and Physiology, Clemson University, SC 29631, USA); and Peanut mottle virus (PeMoV) (Seed Pathology Institute, Flakkejerg, DK-4200 Slagelse, Denmark). The latter two viruses were tested for by immunodot ELISA, and the other viruses by double antibody sandwich-ELISA. Laboratory tests results indicated that the overall virus disease incidence was 15·7%. SMV was the most common virus, with an average of 13·3%, followed by TSWV (5·4%), TRSV (4·2%), TSV (4·1%), PEMV (2·9%), BYMV (2·2%), ToRSV (2·1%), TMV (1·5%), AlMV (1·3%), BCMV (0·8%) and CMV (0·6%). In addition, BlCMV was detected in selected samples with symptoms from Mazandaran and Lorestan, and PeMoV in samples from Golesta. Field symptoms associated with virus infection included mosaic, mottling, crinkle, vein clearing and vein necrosis with SMV; mosaic and mottling with AlMV, BCMV, BlCMV, BYMV, PEMV, PeMoV and TMV; top necrosis in most TRSV and ToRSV-infected plants; and chlorosis and necrosis with TSWV. Plants were frequently infected with more than one virus, however, so it was not possible to correlate specific symptoms with a particular virus. SMV, TSV, TRSV, ToRSV and TSWV have previously been recorded in soybean in Iran (Eskandari, 1978; Rahimian et al ., 1995; Golnaraghi et al. , 2001), and AlMV, BCMV, BYMV, CMV, PEMV-1 and PeMoV have previously been reported from other hosts (Kaiser et al ., 1971; Shahraeen & Bananej, 1995). However, this is the first report of naturally occurring infections of AlMV, BCMV, BlCMV, BYMV, CMV, PEMV-1, PeMoV and TMV in soybean fields in Iran.

Effect of Figaren, an Antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants.

Purification and Partial Characterization of Figaren, an RNase-like Novel Antiviral Protein from Cucumis figarei

An antiviral protein, designated figaren, was purified from leaves of Cucumis figarei and partially characterized. Column chromatography, SDS-polyacrylamide gel electrophoresis and periodic acid-Schiff staining revealed that figaren is a glycoprotein with a molecular weight of 23 kDa. Figaren was stable at pH 2 to 12 and below 90°C. N-terminal amino acid sequencing indicated that figaren contained the conserved region for the S-allele-associated ribonucleases (RNases). In-gel RNase assay showed that figaren digested yeast RNAs. Figaren also digested double-stranded RNAs extracted from Cucumber mosaic virus (CMV)-infected tobacco tissues. Fluorescence in situ hybridization revealed that figaren and RNases (beef pancrease and RNase T1 at 1 μg/ml similarly inhibited CMV infection in cowpea leaves. Figaren and the RNases at 5-500 ng/ml had similar inhibitory effect on local lesion formation by CMV. These data suggest that figaren is a novel RNase-like antiviral protein.