Infected Nicotiana Benthamiana Research Articles

Molecular Identification, Reverse Transcription-Polymerase Chain Reaction Detection, Host Reactions, and Specific Cytopathology of Artichoke yellow ringspot virus Infecting Onion Crops

ABSTRACT An isometric virus ca. 25 nm in diameter with angular contour was isolated from onion plants showing yellow leaf striping and necrotic tips. The virus was mechanically transmitted onto 28 species of indicator plants belonging to five families, viz. Amaranthaceae, Chenopodiaceae, Cucurbitaceae, Leguminosae, and Solanaceae where it causes ring spots, malformations, and/or tip necrosis. Cytopathological studies in infected Nicotiana benthamiana tissues revealed cytoplasmic inclusions resembling those caused by Artichoke yellow ringspot virus (AYRSV), a member of the family Comoviridae. Host range and symptomatology of the onion virus were also similar to AYRSV. A high seed transmission rate (20%) was found in onion. Reverse transcription-polymerase chain reaction using degenerate primers specific for the family Comoviridae allowed amplification of RNA-dependent RNA polymerase sequences, which upon sequence analysis and comparison with AYRSV isolates from Cynara scolymus (AYRSV-AtG) and Vicia faba (AYRSV-F) were highly similar, thus providing evidence that the nepovirus AYRSV is infecting onion in the field.

Biological Relevance of a Stable Biochemical Interaction between the Tombusvirus-Encoded P19 and Short Interfering RNAs

The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R-->W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR-->GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex approximately 21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for approximately 21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.

Identification of regions of the Beet mild curly top virus (family Geminiviridae) capsid protein involved in systemic infection, virion formation and leafhopper transmission

Plant viruses in the genus Curtovirus (family Geminiviridae) are vectored by the beet leafhopper ( Circulifer tenellus) and cause curly top disease in a wide range of dicotyledonous plants. An infectious clone of an isolate of Beet mild curly top virus (BMCTV-[W4]), associated with an outbreak of curly top in pepper and tomato crops, was characterized and used to investigate the role of the capsid protein (CP) in viral biology and pathogenesis. Frameshift mutations were introduced into the overlapping CP and V2 genes, and a series of CP alanine scanning mutations were generated. All mutants replicated in tobacco protoplasts or systemically infected plants, consistent with these gene products not being required for viral DNA replication. The CP frameshift mutant and most C-terminal alanine scanning mutants did not systemically infect Nicotiana benthamiana plants or form detectable virions, and were not leafhopper-transmitted. In contrast, most N-terminal alanine scanning mutants systemically infected N. benthamiana and induced disease symptoms, formed virions and were leafhopper-transmissible; thus, these substitution mutations did not significantly alter the functional properties of this region. One N-terminal mutant (CP49-51) systemically infected N. benthamiana, but did not form detectable virions; whereas another (CP25-28) systemically infected N. benthamiana and formed virions, but was not insect-transmissible. These mutants may reveal regions involved in virus movement through the plant and/or leafhopper vector. Together, these results indicate an important role for virions in systemic infection (long-distance movement) and insect transmission, and strongly suggest that virions are the form in which BMCTV moves, long distance, in the phloem.

First report of Bean common mosaic virus in Western Australia

Bean common mosaic virus (BCMV; genus Potyvirus, family Potyviridae) infects Phaseolus vulgaris crops in many regions of the world. It is transmitted in a nonpersistent manner by aphids and is also readily seed-transmitted (Hongying et al., 2002). The disease it causes decreases crop production. In Australia, BCMV has been reported in New South Wales, Queensland, Tasmania and Victoria, based on serology and amino acid composition (Moghal & Francki, 1976, 1981). However, there is no information about isolates or their origin, and no further confirmation, or any sequence data for BCMV from Australia. It has not been reported in the Northern Territory, South Australia or Western Australia. In July 2004, at Kununurra in the east Kimberly region of Western Australia, P. vulgaris plants with mottle and leaf deformation, severe mosaic, malformation of leaves and pods, downward curling of leaves and reduction in leaf size were observed in the field. Extracts of leaf samples with symptoms tested positive with generic potyvirus monoclonal antibody in ELISA, and infected Chenopodium quinoa, Chenopodium amaranticolor and Nicotiana benthamiana when manually inoculated. For molecular identification, total RNA was isolated from symptomatic leaf tissue using an RNeasy Plant Mini Kit (Qiagen). The RNA samples were tested using RT–PCR and generic potyvirus specific primers that amplify a 1700 bp fragment from the 3′ end of the genome [5′-GTTTTCCCAGTCACGA C(T)15; 5′-GGNAAYAAYAGYGGNCARCC] (Chen et al., 2001). A portion of the PCR product (482 bp) was sequenced at the 3′ end (accession no. AY850005) and the data were compared with other BCMV sequences in GenBank. The isolate shared 98% nucleotide identity with the ‘NL1’ and ‘Type’ strains of BCMV (accession nos AY112735 and U55319). This sequence result provides the first reliable confirmation of BCMV in Australia, and is the first report of its occurrence in Western Australia.

Stachytarpheta leaf curl virus is a novel monopartite begomovirus species

Begomovirus isolates were obtained from Stachytarpheta jamaicensis plants showing leaf curl and chlorosis symptoms collected in the Hainan province of China. The complete sequences of isolates Hn5-4, Hn6-1, Hn30 and Hn34 were determined to be 2748, 2751, 2748 and 2748 nucleotides long, respectively. The complete sequences of the four isolates share more than 94.9% nucleotide sequence identity, but all of them have less than 86% nucleotide sequence identity with other reported begomoviruses. The molecular data show that Hn5-4, Hn6-1, Hn30 and Hn34 are isolates of a distinct begomovirus species, for which the name Stachytarpheta leaf curl virus (StaLCV) is proposed. PCR and Southern blot analyses demonstrate that all the collected field samples are not associated with DNAbeta or DNA-B components. An infectious clone of StaLCV isolate Hn5-4 was constructed, and could efficiently infect Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Lycopersicon esculentum and Petunia hybrida plants, inducing upward leaf roll and vein swelling symptoms. In addition, we illustrate that StaLCV can functionally interact with distinct DNAbeta molecules in plants.

Subcellular localization and immunodetection of movement proteins of olive latent virus 1

Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was observed in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata.

Characterization of Tomato bushy stunt virus newly isolated from nipplefruit (Solanum mammosum) in Japan

Tomato bushy stunt virus (TBSV) was isolated from nipplefruit (Solanum mammosum) in Chiba Prefecture, Japan, with mosaic symptoms, leaf deformation, and stunting. The genomic structure of the TBSV-nipplefruit strain (TBSV-Nf) is identical to that of the TBSV-cherry strain (TBSV-Ch). Transcripts from its full-length cDNA clone without a cap analog at the 5′ terminus infected Nicotiana glutinosa, Nicotiana benthamiana, S. mammosum, Lycopersicon esculentum, Chenopodium amaranticolor, and Chenopodium quinoa, which displayed symptoms identical to those of the parental virus. Although TBSV-Ch systemically infected nipplefruit, systemic symptoms caused by TBSV-Nf were more severe than those by TBSV-Ch. This virus is the first TBSV discovered in Japan and the first TBSV ever isolated from nipplefruit.

POTENTIAL APPLICATION OF VIRUS-INDUCED GENE SILENCING (VIGS) IN FLOWER SENESCENCE STUDIES

Virus-induced gene silencing has been suggested as a powerful and rapid technique for analysis of gene function in plant growth and development (Baulcombe, 1999). Infecting Nicotiana benthamiana with Tobacco Rattle Virus (TRV) containing a fragment of the gene encoding phytoene desaturase, an essential enzyme in carotene synthesis, results in a photo-bleached phenotype in photosynthetic tissues. TRV has several interesting properties that could be utilized in studies of the function of candidate genes in floral senescence; it infects floral tissues, has only mild symptoms, and has a broad host range. We tested the effect of TRV containing PDS fragments on a range of host plants. In most cases, infection resulted in no apparent phenotype or local inoculation effects. In petunia, after a lag of several weeks, the characteristic photo-bleaching of upper portions of the plant indicated movement and silencing of the virus and of PDS. In preliminary tests with a TRV/CHS construct, the typical effects of post-transcriptional gene silencing of CHS in petunia – symmetrical and asymmetrical white patches on the dark purple petals – indicated the ability to use the system in petunia studies.

First Report of Zucchini yellow mosaic virus in Cucumber (Cucumis sativus) in India.

In August of 2002, cucumber (Cucumis sativus L. cv. Himangi) plants grown in commercial fields in Pune, western Maharashtra, India, exhibited chlorotic spots, veinal chlorosis, mosaic, blister formation and shoestring symptoms on leaves, stunted growth, and distortion of fruits. Incidence of virus infection in the fields varied from 25 to 38%. Crude sap extracted from infected cucumber leaf samples was inoculated mechanically onto cucumber and indicator host plants. The inoculated glasshouse-grown cucumber plants showed virus symptoms similar to those observed in the field. The virus produced chlorotic local lesions on Chenopodium amaranticolor and chlorotic spots followed by veinal chlorosis, mosaic, vein banding, and leaf distortion on Citrullus lanatus, C. melo, C. sativus, Cucurbita maxima, Cucurbita pepo, Luffa acutangula, and Trichosanthes anguina. The virus did not infect Nicotiana benthamiana, N. glutinosa, and N. tabacum cv. White Burley (1,2). Cucumber and indicator plants were tested using direct antigen coating enzyme-linked immunosorbent assays. A positive reaction was obtained with monoclonal antiserum to Zucchini yellow mosaic virus (ZYMV) but not with antisera to Papaya ringspot virus-P, Cucumber mosaic virus, and Watermelon mosaic virus (Agdia, Inc., Elkhart, IN). The disease was observed again in the field during July to September 2003. Natural infection of zucchini (Cucurbita pepo) by ZYMV has been reported previously from India (3). To our knowledge, this is the first report of ZYMV in cucumber in India.

Functional analysis of the final steps of the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway to isoprenoids in plants using virus-induced gene silencing.

Isoprenoid biosynthesis in plant plastids occurs via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. We used tobacco rattle virus (TRV) to posttranscriptionally silence the expression of the last two enzymes of this pathway, the IspG-encoded (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) and the IspH-encoded isopentenyl/dimethylallyl diphosphate synthase (IDDS), as well as isopentenyl/dimethylallyl diphosphate isomerase (IDI), the enzyme that interconverts IPP and DMAPP. TRV-IspG and TRV-IspH infected Nicotiana benthamiana plants had albino leaves that contained less than 4% of the chlorophyll and carotenoid pigments of control leaves. We applied [(13)C]DXP and [(14)C]DXP to silenced leaves and found that 2-C-methyl-d-erythritol 2,4-cyclodiphosphate accumulated in plants blocked at HDS while DXP, (E)-4-hydroxy-3-methylbut-2-enyl phosphate and (E)-2-methylbut-2-ene-1,4-diol accumulated in IDDS-blocked plants. Albino leaves from IspG- and IspH-silenced plants displayed a disorganized palisade mesophyll, reduced cuticle, fewer plastids, and disrupted thylakoid membranes. These findings demonstrate the participation of HDS and IDDS in the DXP pathway in plants, and support the view that plastid isoprenoid biosynthesis is metabolically and physically segregated from the mevalonate pathway. IDI-silenced plants had mottled white-pale green leaves with disrupted tissue and plastid structure, and showed an 80% reduction in pigments compared to controls. IPP pyrophosphatase activity was higher in chloroplasts isolated from IDI-silenced plants than in control plant chloroplasts. We suggest that a low level of isoprenoid biosynthesis via the DXP pathway can occur without IDI but that this enzyme is required for full function of the DXP pathway.

Expression of the B subunit of the heat-labile enterotoxin of Escherichia coli in tobacco mosaic virus-infected Nicotiana benthamiana plants and its characterization as mucosal immunogen and adjuvant

We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coliin tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 μg rLTB pentamers per gram fresh plant material and was capable of binding G M1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 μg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.

A Virus Related to Clover Yellow Mosaic Virus Found East of the Mississippi River in Verbena canadensis in Florida.

In September 2002, several unthrifty Verbena canadensis 'Homestead purple' plants were received at the Division of Plant Industry in Gainesville, FL. Symptoms included very subtle yellowing and distortion or stunting of the younger leaves, symptoms that could be overlooked as a nutritional problem. Symptomatic leaves were ground in phosphate buffer and mechanically inoculated to a variety of plants that included Antirrhinum majus, Chenopodium amaranticolor, Datura stramonium, Gomphrena globosa, Pisum sativum, Trifolium pratense, T. repens, Vicia faba, and Vigna unguiculata. These hosts showed symptoms similar to those described for infection by Clover yellow mosaic virus (ClYMV) (3). In addition, the virus systemically infected Arachis hypogaea, Catharanthus roseus, C. quinoa, Nicotiana benthamiana, and healthy seed-grown Verbena × hybrida. No symptoms were seen in inoculated Zinnia elegans, N. glutinosa, N. clevelandii, Lycopersicon esculentum, Cucumis sativus, or Capsicum annuum. However, back inoculations of Cucumis sativus to C. amaranticolor gave typical local and systemic symptoms. Microscopic examination of leaf strips of infected V. faba revealed banded inclusions typical of the genus Potexvirus (1). Reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers for the genus Potexvirus (2) produced a 750-bp product that is the expected product for the genus Potexvirus. The RT-PCR product was cloned in pGem-T easy (Promega, Madison, WI) and sequenced. BLAST ( http://www.ncbi.nlm.nih.gov/ BLAST/ ) analysis of the sequence showed an 82% identity at the nucleotide level with the RNA polymerase gene of ClYMV. Leaf extracts of the original verbena, several inoculated hosts, and a known sample of ClYMV in N. benthamiana were tested in sodium dodecyl sulfate immunodiffusion (4) against antiserum to the degraded capsid protein of ClYMV. A reaction of identity was seen with infected samples but not with samples for comparable virus-free plants. To our knowledge, this is the first time this virus has been found in the eastern United States. It is not known how or if the presence of this noninsect transmitted virus in an ornamental will affect the agricultural, forage, or ornamental industries in the east or how widely distributed Verbena sp. infected with this virus may be at this time.

Expression of active human epidermal growth factor (hEGF) in tobacco plants by integrative and non-integrative systems

Human epidermal growth factor (hEGF) was expressed in Nicotiana plants using integrative and non-integrative systems. Nuclear transformation was carried out using genetic constructs that allow hEGF accumulation into the cytoplasmic or apoplastic spaces. Protein level did not exceed 0.00001% of total soluble protein in the cytoplasm, but reached values of up to 0.11% when hEGF was targeted to the apoplast. In addition, cytoplasmic and apoplastic hEGF versions were cloned into a viral vector derived from potato virus X. Transcripts from recombinant viruses were used to infect Nicotiana benthamiana and Nicotiana tabacum plants. While the recombinant protein was barely detectable in the case of the cytoplasmic version, it reached levels of up to 0.015% of total soluble proteins in plants infected with the apoplastic version. Extracts from transgenic plants exhibiting the highest hEGF accumulation and from plants infected with the viral apoplastic version were tested for biological activity in cumulus cells expansion assays. hEGF containing extracts showed biological activity comparable to commercial hEGF. Finally, radioreceptor binding assays showed that tobacco-expressed hEGF binds to its receptor with the same affinity as commercial hEGF. These data suggest that hEGF accumulation can be significantly increased by implementing adequate exporting strategies.

Individualized human scFv vaccines produced in plants: humoral anti-idiotype responses in vaccinated mice confirm relevance to the tumor Ig

We have developed a method for rapidly producing in plants the idiotype regions of the tumor-specific Ig as single-chain Fv (scFv) proteins for use in the treatment of non-Hodgkin's lymphoma. Variable region gene sequences were generated from either a tumor hybridoma or human tumor biopsy cells, and idiotype domains were joined by a novel linker and cloned into a modified tobacco mosaic virus (TMV) vector designed to secrete the scFv protein in infected Nicotiana benthamiana plants. Thirty-eight out of 44 human scFv proteins showed Coomassie visible material in crude secretory (interstitial fluid, IF) extracts, 21 of those between 100 and 800 μg/ml. Eight of these proteins were tested for appropriate idiotype responses in vaccinated mice. In all eight cases, anti-idiotype immune responses were induced with minimal cross reactivity to irrelevant Ig or scFv proteins. Four out of four anti-scFv sera were also shown to recognize the Ig on human tumor cells by flow cytometry analysis.

A naturally occurring recombinant DNA-A of a typical bipartite begomovirus does not require the cognate DNA-B to infect Nicotiana benthamiana systemically

Species of the genus Begomovirus (family Geminiviridae) found in the western hemisphere typically have a bipartite genome that consists of two 2.6 kb DNA genomic components, DNA-A and DNA-B. We have identified and cloned genomic components of a new tomato-infecting begomovirus from Brazil, for which the name Tomato crinkle leaf yellows virus (TCrLYV) is proposed, and a DNA-A variant of Tomato chlorotic mottle virus (ToCMV-[MG-Bt1]). Sequence analysis revealed that TCrLYV was most closely related to ToCMV, although it was sufficiently divergent to be considered a distinct virus species. Furthermore, these closely related viruses induce distinguishable symptoms in tomato plants. With respect to ToCMV-[MG-Bt1] DNA-A, evidence is presented that suggests a recombinant origin. It possesses a hybrid genome on which the replication compatible module (AC1 and replication origin) was probably donated by ToCMV-[BA-Se1] and the remaining sequences appear to have originated from Tomato rugose mosaic virus (ToRMV). Despite the high degree of sequence conservation with its predecessors, ToCMV-[MG-Bt1] differs significantly in its biological properties. Although ToCMV-[MG-Bt1] DNA-A did not infect tomato plants, it systemically infected Nicotiana benthamiana, induced symptoms of mottling and accumulated viral DNA in the apical leaves in the absence of a cognate DNA-B. The modular rearrangement that resulted in ToCMV-[MG-Bt1] DNA-A may have provided this virus with a more aggressive nature. Our results further support the notion that interspecies recombination may play a significant role in geminivirus diversity and their emergence as agriculturally important pathogens.

Host DNA replication is induced by geminivirus infection of differentiated plant cells.

The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.

MOLECULAR CHARACTERIZATION OF A TOMBUSVIRUS ISOLATED FROM DISEASED PEAR TREES IN SOUTHERN ITALY

A tombusvirus first isolated in 1991 in Apulia (southern Italy) from pear trees (Pyrus communis L.) showing reduced growth, reddish discolourations and premature shedding of the leaves, was again recovered from symptomatic plants and characterized. Agarose gel analysis of extracts from systemically infected Nicotiana benthamiana plants showed the presence of two major and one minor RNA species with a size compatible with that of genomic RNA and of monomeric and dimeric defective interfering (DI) RNAs of tombusviruses. RNA was extracted from purified virus particles. All these RNA species were cloned, sequenced and shown to have a typical tombusviral structural organization. The most prominent cytopathological feature of N. benthamiana infected cells was the presence of peroxisome-derived multivesicular bodies. In immunodiffusion tests this virus proved to be serologically related with some tombusviruses, the closest being Grapevine Algerian latent virus (GALV), but showed wide molecular differences with all sequenced members of the genus. This virus was successfully transmitted to pear seedlings and rooted cuttings that were still symptomless five months after inoculation. Thus, it was given the provisional name of Pear latent virus (PLV).

First Report of Zucchini yellow mosaic virus on Hollyhock (Althaea rosea)

This study was conducted to determine the causal virus that naturally infected hollyhock (Althaea rosea) plant showing mild mosaic symptom in 1999. Flexuous virus particles were found in the cytoplasm of plant tissue from infected hollyhock under transmissible electron microscopy. A virus from the genus Potyvirus under the family Potyviridae was isolated and was maintained on Chenopodium quinoa for three passages. Chlorotic local legions were used to inoculate 20 species of indicator plants. The virus infected all the tested cucurbit plants, but failed to infect Nicotiana benthamiana. Based on the host range test and RT-PCR analysis, the potyvirus was identified as a strain of Zucchini yellow mosaic virus-A (ZYMV-A), one of the major pathogens of cucurbits. Infectivity analysis showed that ZYMV-A induced faster systemic symptom than ZYMV-Cu on squash and other cucurbit plants, suggesting that ZYMV-A was a more severe strain. To better characterize ZYMV-A, Western blot assay was carried rout to the coat protein (CP) of the virus using ZYMV-specific antiserum with ZYMV-Cu and other potyviruses. The CP of the virus reacted strongly with the antiserum against ZYMV, and other tested antisera did not react with the CP of ZYMV-A. Results strongly suggest that the potyvirus infecting hollyhock was a novel strain of ZYMV. This is the first report on ZYMV as the causal virus infecting hollyhock in Korea.

Occurrence of Lily mottle virus in Escarole.

Lily mottle virus (LMoV), genus Potyvirus, an important virus of lily that also causes flower-breaking in tulip (1), is considered to have a natural host range restricted to the family Liliaceae. In 1996, escarole (Cichorium endivia L. var. latifolium LAM) plants growing in fields close to Torino, Italy, and showing mosaic and necrotic spots on outer leaves were infected by a potyvirus related to LMoV. The virus was identified by immunoelectron microscopy (IEM) done on experimentally infected Nicotiana benthamiana and Chenopodium quinoa. The virus isolated from escarole (LMoV-E) had an experimental host range similar to that of lily isolates of LMoV, although species within the Liliaceae were not tested. LMoV-E systemically infected all nine escarole cultivars and six of seven endive cultivars (C. endivia L. var. crispum LAM) but did not infect any of six lettuce and two chicory cultivars (C. intybus L. var. foliosum HEGI). Symptoms ranged from mild to severe mosaic and were generally more severe on escarole than on endive. Some of the same escarole, endive, and lettuce cultivars were inoculated with a typical LMoV isolate from lily (Le97/49, from A. F. L. M. Derks, the Netherlands), which induced mild systemic infections in only one escarole and one endive cultivar. Both cultivars were also susceptible to LMoV-E. LMoV-E was purified from N. benthamiana, and an antiserum was prepared. IEM decoration titer experiments were done with LMoV-E and four other LMoV isolates from Liliaceae and their homologous antisera. Heterologous titers ranging from identity to serological differentiation index values of 2 to 4 were obtained, confirming the identity of the escarole isolate as a LMoV strain and indicating considerable serological variability among LMoV isolates. In a field survey of endive and escarole crops in 1998, in the area where LMoV-E was first identified, the virus was again detected by enzyme-linked immunosorbent assay in 4 of 80 escarole plants tested. LMoV-E appears to be a LMoV strain particularly adapted to escarole. To our knowledge, this is the first report of LMoV identified in a naturally infected host outside monocotyledonous plants. Reference: (1) E. L. Dekker et al. J. Gen. Virol. 74:881, 1993.

First Report of Pepino mosaic virus on Tomato in Spain.

At the beginning of 2000, a damaging disease developed on protected tomato (Lycopersicon esculentum) crops grown in polyethylene greenhouses in different regions of Spain. Production losses were estimated at 15 to 80%. The tomato plants showed a variety of symptoms. The most common symptoms were leaf distortion, chlorosis, and mosaic. Some plants showed a dark green mosaic and bubbling of the leaf surface. Green striations were also observed on the stem and sepals. Most of the diseased plants had discolored fruits. Symptoms decreased as environmental temperature increased. The involvement of Pepino mosaic virus (PepMV) was suspected. To identify the etiological agent, ≈500 symptomatic tomato plants were collected from several locations in Alicante, Murcia, Almeria and the Canary Islands. Flexuous viral particles 510 nm long were observed by transmission electron microscopy, suggesting the presence of a potexvirus in the tissue extracts analyzed. All samples were tested by ELISA (enzyme-linked immunosorbent assay), using polyclonal antibodies to Narcissus mosaic virus (Adgen, Auchincriuve, Scotland), a virus serologically related to PepMV, and two antisera specific to PepMV (Adgen, Scotland and DMSZ, Braunschweig, Germany). PepMV was detected in 35% of the samples. Like PepMV, the virus infected (as confirmed by ELISA) greenhouse-grown Datura stramonium, Nicandra physalodes, Nicotiana benthamiana, N. clevelandii, Solanum tuberosum, and Vigna sinensis and did not infect Capsicum anuum, Cucumis sativus, Chenopodium amaranticolor, C. quinoa, Petunia × hybrida, Phaseolus vulgaris, Physalis floridana, N. glutinosa, N. rustica, or N. tabacum. The virus did infect Gomphrena globosa, which normally is not infected by PepMV. The first report of PepMV was on pepino (Solanum muricatum) in Peru in 1974 (1), but this virus has been recently reported in the Netherlands, England, Germany, and France on protected tomato crops (2). To our knowledge, this is the first report of PepMV in Spain, including the Canary Islands.