Rapid and low-cost diagnosis of <Emphasis Type="Italic">Japanese yam mosaic virus</Emphasis> infection in Chinese yam (<Emphasis Type="Italic">Dioscorea polystachya</Emphasis>) leaves by a print-capture RT-PCR

A rapid diagnostic method for Japanese yam mosaic virus (JYMV) infection in Chinese yam (Dioscorea polystachya) by a print-capture RT-PCR was developed. The cut surface of a rolled leaf of a JYMV-infected Chinese yam was stamped onto a nitrocellulose membrane. The clipped membrane was boiled in eluting solution to elute the RNA for use as the template in RT-nested PCR. Although JYMV RNA was detectable in most infected plants in the first PCR of the print-capture RT-PCR, it was detected from all samples in the nested PCR. The tissue-printed membranes could be preserved for at least 3 months at 4?°C for JYMV detection.     

First report of <Emphasis Type="Italic">Bean common mosaic virus</Emphasis> in yam bean [<Emphasis Type="Italic">Pachyrhizus erosus</Emphasis> (L.) Urban] in Indonesia

Severe mosaic with leaf malformation and green vein banding was observed on yam bean in West and Central Java, Indonesia. Virions of the causal virus were flexuous filaments, about 700 nm in length, with a coat protein of 30 kDa. The virus was transmitted by mechanical inoculation and by aphids in a nonpersistent manner. The nucleotide sequence of the coat protein gene had the highest identity with that of Bean common mosaic virus (BCMV, genus Potyvirus) isolate VN/BB2-5. Based on demarcation criteria, including the genome sequence and host range, we tentatively designate this isolate as BCMV-IYbn (Indonesian yam bean). The nucleotide sequence reported is available in the DDBJ/EMBL/GenBank databases under accession number AB289438.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

Partial characterisation of a novel <Emphasis Type="Italic">Tobamovirus</Emphasis> infecting <Emphasis Type="Italic">Actinidia chinensis</Emphasis> and <Emphasis Type="Italic">A. deliciosa</Emphasis> (Actinidiaceae) from China

Actinidia chinensis and A. deliciosa plants from China, showing a range of symptoms, including vein clearing, interveinal mottling, mosaics and chlorotic ring spots, were found to contain ~300 nm rod-shaped virus particles. The virus was mechanically transmitted to several herbaceous indicators causing systemic infections in Nicotiana benthamiana, N. clevelandii, and N. occidentalis, and local lesions in Chenopodium quinoa. Systemically- infected leaves reacted with a Tobacco mosaic virus polyclonal antibody in indirect ELISA. PCR using generic and specific Tobamovirus primers produced a 1,526 bp sequence spanning the coat protein (CP), movement protein (MP), and partial RNA replicase genes which showed a maximum nucleotide identity (88%) with Turnip vein clearing virus and Penstemon ringspot virus. However, when the CP sequence alone was considered the highest CP sequence identity (96% nt and 98% aa) was to Ribgrass mosaic virus strain Kons 1105. The morphological, transmission, serological and molecular properties indicate that the virus is a member of subgroup 3 of the genus Tobamovirus.     

<Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> isolated from Amazon lily (<Emphasis Type="Italic">Eucharis grandiflora</Emphasis>)

Cucumber mosaic virus (CMV) was isolated from a mosaic diseased plant of Eucharis grandiflora. The virus caused mosaic symptoms on leaves and slight distortion of flower petals in E. grandiflora by either mechanical or aphid inoculation. The virus was identified as a strain of CMV subgroup I from its biological and serological characteristics.     

Molecular characterisation of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> isolates from Brassicaceae weeds

Eight provinces of Iran were surveyed during 2003–2008 to find Brassicaceae reservoir weed hosts of Turnip mosaic virus (TuMV). A total of 532 weed samples were collected from plants with virus-like symptoms. The samples were tested for the presence of TuMV by enzyme-linked immunosorbent assay using specific antibodies. Among those tested, 340 samples (64%) were found to be infected with TuMV. Rapistrum rugosum, Sisymberium loeselii, S. irio and Hirschfeldia incana were identified as the Brassicaceae weed hosts of TuMV, and the former two plant species were found to be the most important weed hosts for the virus in Iran. The full-length sequences of the genomic RNAs of IRN TRa6 and IRN SS5 isolates from R. rugosum and S. loeselii were determined. No evidence of recombination was found in both isolates using different recombination-detecting programmes. Phylogenetic analyses of the weed isolates with representative isolates from the world showed that the IRN TRa6 and IRN SS5 isolates fell into an ancestral basal-Brassica group. This study shows for the first time the wide distribution and phylogenetic relationships of TuMV from weeds in the mid-Eurasia of Iran.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.     

<Emphasis Type="Italic">Turnip yellow mosaic virus</Emphasis> isolated from Chinese cabbage in Japan

A virus that caused a distinct yellow mosaic was isolated in Okayama, Japan from Chinese cabbage (Brassica rapa L., Pekinensis group). The virus, with spherical particles ca. 28 nm in diameter, was mechanically transmissible only to cruciferous species. From the host range, characteristic morphology of virus particles, serology and sequence analysis of coat protein gene, the causal virus was identified as Turnip yellow mosaic virus (TYMV). Seed transmission of TYMV at 0–2.2% in Chinese cabbage was confirmed. This report is the first of TYMV from Chinese cabbage and in Japan. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases as accessions AB358971 and AB358972.     

Improved PCR detection of potyviruses in <Emphasis Type="Italic">Allium</Emphasis> species

Protocols for producing virus-free Allium plants require an indexing system that is more sensitive than DAS-ELISA and can detect low virus concentrations in infected plants. In the present work, degenerate primers were designed and a one-step IC-RT-PCR protocol was developed to differentiate between Leek yellow stripe virus (LYSV) and Onion yellow dwarf virus (OYDV) in single and mixed infections in several Allium spp. A 566-bp band was observed for LYSV, a 489-bp band for OYDV in single infections, and two bands of the same sizes in mixed infections in different species of Alliaceae. A 508-bp band of Shallot yellow stripe virus and a 594-bp band of Turnip mosaic virus were also amplified with the same primers. RT-nested-PCR was also conducted directly in microtitre plate wells after negative or questionable reactions were produced in an ELISA experiment. The detection limit of the DAS-ELISA for LYSV was 16.5–27.3 ng ml⁻¹. The RT-nested-PCR done after DAS-ELISA was 10² times more sensitive than the DAS-ELISA alone. In parallel, an IC-RT-nested-PCR in microcentrifuge tubes was 10⁴ times more sensitive than the DAS-ELISA. The DAS-ELISA-RT-nested-PCR enables the initial screening of samples by DAS-ELISA to eliminate a high percentage of virus-positive plants, considerably reducing the number of plants to analyze further by RT-PCR.     

The coat protein of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> L<Subscript>11</Subscript>Y is associated with virus-induced chlorosis on infected tobacco plants

The L₁₁Y strain of Tomato mosaic virus (ToMV) causes severe chlorosis on infected tobacco leaves. Sequencing analysis for the genome showed that L₁₁Y contained multiple nucleotide changes and that some led to amino acid substitutions, when compared with that of the common L strain of ToMV. The chimeric virus, which has the CP of L₁₁Y in the context of the L strain RNA genome, caused severe chlorosis on infected tobacco plants, suggesting that the CP of L₁₁Y containing three amino acid changes (E33S, A86T and E97K) was the determinant of the chlorosis. Two of these amino acid changes (A86T and E97K) were associated with the induction of chlorosis when present together in the CP. Severe destruction and deformation of chloroplasts and the formation of discrete dark-staining materials adjacent to chloroplasts were observed with electron microscopy in L₁₁Y-infected plants. Fewer virus particles accumulated in the cytoplasm of L₁₁Y-infected plant cells. The level of accumulation of CP subgenomic RNA and CP in the infected protoplasts was similar between L and L₁₁Y. Fewer virus particles accumulated in L₁₁Y-infected protoplasts, and many of them were shorter-than-full-length. The nucleotide sequence data reported is available in DDBJ/EMBL/GenBank databases as accession AB355139.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454     

First Report of <Emphasis Type="Italic">Pepper mottle virus</Emphasis> on <Emphasis Type="Italic">Capsicum annuum</Emphasis> in Japan

Pepper mottle virus, genus Potyvirus, was first identified in Japan based on particle morphology, host range, aphid transmission, and molecular classification using the nucleotide sequence of the coat protein gene and 3-untranslated region.     

Genetic characterization of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolates from Belgian greenhouse tomatoes reveals genetic recombination

Over a period of a few years, Pepino mosaic virus (PepMV) has become one of the most important viral diseases in tomato production worldwide. Infection by PepMV can cause a broad range of symptoms on tomato plants, often leading to significant financial losses. At present, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described, three of which (EU, LP and US2) have been reported in Europe. Thus far, no correlation has been found between different PepMV genotypes and the symptoms expressed in infected plants. In this paper, the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to symptom development in tomato crops. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate between the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype and the CH2 genotype, within tomato production in Belgium. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in mixed infections under natural conditions. This may possibly result in the generation of viral variants with increased aggressiveness.     

First report of a <Emphasis Type="Italic">Grapevine Algerian latent virus</Emphasis> disease on statice plants (<Emphasis Type="Italic">Limonium sinuatum</Emphasis>) in Japan

A viral disease was found in Nagano Prefecture, Japan, on statice (Limonium sinuatum) with chlorotic leaf spot, necrotic stunt, and dwarfing. Spherical virus particles 30 nm in diameter were isolated from infected plants and statice seedlings and caused identical symptoms 4 weeks after mechanical inoculation. Nucleotide and deduced amino acid sequences of the coat protein showed 98% and 98.7% identities with those of Grapevine Algerian latent virus (GALV) nipplefruit strain. This is the first report in Japan of a viral disease on statice caused by GALV. The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank databases under accession AB461854.     

Preservation of <Emphasis Type="Italic">Alfalfa mosaic virus</Emphasis> by freezing and freeze-drying and similarities to Cucumoviruses

Alfalfa mosaic virus (AMV) belongs to the genus Alfamovirus of the family Bromoviridae, for which the virions are stabilized by dominant protein–RNA interactions. The infectivity of purified AMV preparations stored frozen at −20°C decreased to 10–20% in 2 years. In addition, the virion peak profiles after sucrose density gradient centrifugation (SDGC) was reduced to a single, broad peak as a result of virus particle degradation, and the peaks for the extracted virion RNA decreased. However, additives such as 0.5% peptone or 2.5% sucrose were markedly protective such that infectivity and the SDGC profiles of the virus particles and virion RNA remained essentially unchanged after 5–8 years of freezing. Infectivity of the purified AMV decreased to c. 50%, and virus particles deteriorated immediately after freeze-drying. The addition of 1.0–7.5% sucrose suppressed alterations in infectivity, particle morphology and virion RNA after freeze-drying and other preservation processes. The characteristics of AMV preservation were similar to those reported in a previous study on cucumoviruses. Consequently, viruses belonging to the Bromoviridae may preserve well with sucrose in conjunction with freezing or freeze-drying.     

The genetic structure of populations of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> in Kyushu and central Honshu,Japan

The genetic structure of the populations of Turnip mosaic virus in Kyushu and central Honshu, Japan was assessed. The host specificity of isolates was determined, and their gene sequences compared utilizing a population genetic approach. Phylogenetic analysis of partial sequences revealed that 32 of 49 Honshu isolates (65%) collected during 1997–2001 belonged to the basal-BR group as did 23 of 64 isolates from Kyushu. All these basal-BR isolates infected both Brassica and Raphanus plants. However, analyses of the positions of recombination sites in five regions of the genome (one third of the full sequence) showed that at least four intra-lineage recombinants were present in these populations. These analyses showed that Kyushu and Honshu shared none of these subpopulations, and genetically distinct basal-BR populations were present in the two districts. We conclude that different basal-BR subpopulations had expanded into those districts. The nucleotide sequences are deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB267281-AB267376.