<Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> Is Transmissible from Tomato to Tomato by Pollinating Bumblebees

Tobacco mosaic virus (TMV) was detected by ELISA, electron microscopy and/or bioassay from bumblebee (Bumbus terrestris), pollen clumps, nest materials and bee-visited anthers of flowers from greenhouses in which tomatoes had been pollinated by bees and were severely infected with TMV. Experimental bee-mediated transmission of TMV in greenhouse tomatoes demonstrated that the bumblebees transported TMV from plant to plant and that they spread the virus in greenhouses. This is the first report describing TMV transmission by bumblebees. Received 11 August 1999/ Accepted in revised form 30 September 1999     

Movement of <Emphasis Type="Italic">Cucumber Mosaic Virus</Emphasis> Is Restricted at the Interface between Mesophyll and Phloem Pathway in <Emphasis Type="Italic">Cucumis figarei</Emphasis>

Viral movement in the leaf tissues of a resistant host, Cucumis figarei, inoculated with the pepo strain of Cucumber mosaic virus (CMV) and incubated at 24°C or 36°C was investigated by fluorescence in situ hybridization (FISH), leaf-press blotting, tissue printing and immunogold-silver staining techniques. Observation by FISH revealed that at 24°C most infection sites with CMV at 0.01 mg/ml or 0.1 mg/ml were limited to a single cell during the incubation period, that the number of infection sites increased from 24hpi (hours post inoculation) to 80 hpi in the leaves inoculated with CMV at 0.5 mg/ml, and that the size as well as the number of infection sites rapidly increased with time in the leaves inoculated with CMV at 2.0 mg/ml. These results suggested that one factor for the resistance of C. figarei at 24°C might be an inhibition of viral movement in and out of the infection sites. Leaf-press blotting and tissue blotting indicated that CMV remained in the infection sites at 24°C, whereas it spread from the inoculated leaves to other parts of the plants through vascular systems at 36°C. Immunogold-silver staining demonstrated that at 24°C CMV infected bundle sheath (BS) cells in minor veins, whereas at 36°C it invaded not only BS cells, but also phloem parenchyma (PP)/ companion cell (CC) or PP/intermediary cell (IC) complexes in minor veins in the regions with chlorotic symptoms. These results indicated that at 24°C CMV had difficulty in passing through the interface between BS and PP/CC or PP/ IC complexes and that viral entry from mesophyll to the phloem pathway was inhibited in the inoculated leaves. Received 26 August 1999/ Accepted in revised form 14 December 1999     

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.     

<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.     

<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.     

<Emphasis Type="Italic">Turnip ringspot virus</Emphasis> recognised on Chinese cabbage in Russia

The nucleotide sequence of the 3′-terminal part of the RNA1 genome segment of the M12 isolate of comovirus Turnip ringspot virus (TuRSV) was established. This isolate originated in 1989 in Moscow (Russia) from Chinese cabbage with Radish mosaic virus-like symptoms. Comparison of the M12 RNA polymerase amino acid sequence with that of Radish mosaic virus (RaMV) revealed significant differences; these proteins are of different length and are only about 75% identical. On the other hand, the amino acid sequence of the M12 RNA polymerase was more than 94% identical with that of TuRSV recently described in Toledo (USA). We conclude that TuRSV occurs in Europe as well as in America and probably represents a new species of the genus Comovirus.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

Phylogeny of Egyptian isolates of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> (CMV) and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) infecting <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Four Cucumber mosaic virus (CMV) (CMV-HM 1–4) and nine Tomato mosaic virus (ToMV) (ToMV AH 1–9) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98–99%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99–1 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate.     

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.     

Simultaneous detection of <Emphasis Type="Italic">Japanese yam mosaic virus</Emphasis> and <Emphasis Type="Italic">Yam mild mosaic virus</Emphasis> from yam leaves using a tube capture reverse transcription-polymerase chain reaction assay

To detect Japanese yam mosaic virus (JYMV) and Yam mild mosaic virus (YMMV) in yam plants in Japan, we developed a duplex RT-PCR assay consisting of a tube-capture procedure followed by one-step RT-PCR with two primer pairs. A 241-bp fragment of the coat protein region of JYMV and a 174-bp fragment of the nuclear inclusion protein b region of YMMV were amplified, thus identifying the two viruses from yam plants cultivated in Yamaguchi Prefecture in 2007. All water yam plants examined were infected with YMMV alone. All the Japanese yam and Chinese yam plants were infected with either JYMV alone or both JYMV and YMMV, suggesting that YMMV and JYMV are prevalent among field-grown yam plants.     

Resistance of <Emphasis Type="Italic">Solanum</Emphasis> species to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and its vector <Emphasis Type="Italic">Myzus persicae</Emphasis>

Sixty-nine tomato genotypes representing nine Solanum species were evaluated for resistance to Cucumber mosaic virus (CMV) subgroup IA and its aphid vector Myzus persicae. Resistance was assessed by visual scoring of symptoms in the field under natural conditions, and in the greenhouse by artificial inoculations through aphid M. persicae and mechanical transmissions in the year 2007 and 2009. Considerable variation in responses was observed among the evaluation methods used. Field evaluations were found liable to errors as different levels were observed for the same genotypes in the different years, however mechanical inoculation was found to be the most useful in identifying CMV subgroup IA resistance, in contrast aphid transmission was most useful in identifying insect transmission resistance. All genotypes observed as highly resistant to CMV subgroup IA in the field or through vector transmission became systemically infected through mechanical inoculations. Using mechanical inoculation, six genotypes (TMS-1 of S. lycopersicum, LA1963 and L06049 of S. chilense, LA1353, L06145 and L06223 of S. habrochaites) were found resistant and another six (L06188 and L06238 of S. neorickii, L06219 of S. habrochaites, L05763, L05776 and L06240 of S. pennellii) were found tolerant showing mild symptoms with severity index (SI) ranging 1-2 and with delayed disease development after a latent period (LP) of 18–30 days. However, these genotypes were found to be resistant to highly resistant in the field and through inoculation by M. persicae; and they also supported low population levels of M. persicae except TMS-1. Another nine genotypes (LA2184 of S. pimpinellifolium L., LA2727 of S. neorickii, LA0111, L06221, L06127 and L06231 of S. peruvianum L., LA1306, L06057 and L06208 of S. chmielewskii) showing a susceptible response after mechanical inoculation were highly resistant, resistant and tolerant after M. persicae transmission. The resistant genotypes, identified in the present study can be exploited in the breeding programmes aimed at developing tomato varieties resistant to CMV subgroup IA and broadening the genetic base of CMV-resistant germplasm. The differences observed between mechanical and aphid transmission suggests that one should consider both evaluation methods for tomato germplasm screening against CMV subgroup IA.     

Evidence of <Emphasis Type="Italic">olive mild mosaic virus</Emphasis> transmission by <Emphasis Type="Italic">Olpidium brassicae</Emphasis>

Transmission of three strains of OMMV by an Olpidium sp. was evaluated and compared. The three strains were 1) an OMMV wild type (WT) recovered from olive trees, 2) an OMMV variant (L11) obtained after 15 serial passages of single local lesions induced in Chenopodium murale plants, and 3) a construct OMMV/OMMVL11 in which the coat protein (CP) gene replaced that of the wild type. A single-sporangial culture derived from Chinese cabbage (Brassica pekinensis) used as a bait plant grown in soil of an olive orchard, was identified as Olpidium brassicae based on the size and sequence of the generated amplicon in PCR specific tests. Each of the three virus strains was soil transmitted to cabbage roots in the absence of the fungus at similar rates of 30 to 40%. Separate plant inoculation by O. brassicae zoospores incubated with each viral strain resulted in enhanced transmission of OMMV, reaching 86% of infection whereas that of the other two strains remained practically unaffected at ca. 34%. Binding assays showed that the amount of virus bound to zoospores, estimated spectrophotometrically, was 7% in the case of OMMV, and practically nil in the case of the other two viral strains. Substitution of the coat protein (CP) gene of OMMV by that of the OMMV L11 strain, drastically reduced viral transmissibility in the presence of zoospores to the level of that observed in their absence. Our data shows that OMMV soil transmission is greatly enhanced by O. brassicae zoospores and that the viral CP plays a significant role in this process, most likely by facilitating virus binding and later entrance into the host plant roots.     

An infectious cDNA clone of a radish-infecting <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> strain

Turnip Mosaic Virus (TuMV) is an economically important potyvirus for which hundreds of hosts have been reported, thus making it a rather exceptional case in the genus. Several viral infectious clones have been generated over the years, which have been useful in deciphering the viral elements involved in the interactions of this virus with the host plant, such as different forms of resistance, gene silencing suppression, host range or host developmental alterations. However, all infectious clones obtained so far correspond to viral isolates within the same phylogenetic cluster, a circumstance biasing our understanding of the peculiarities of this potyvirus. In particular, members of one viral cluster of radish-infecting isolates have been especially reluctant to be copied into infectious clones. This paper reports the construction of an infectious clone of the TuMV isolate JPN 1, belonging to this cluster. The infectious clone maintains all the distinctive biological properties previously described for this viral isolate. The availability of this infectious clone opens the door to many additional studies on the virus, which should allow a deeper understanding of the differential responses to different strains of TuMV in several different hosts.     

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.     

Isolation of Molecular Markers Linked to the <Emphasis Type="Italic">Cry</Emphasis> Locus Conferring Resistance to <Emphasis Type="Italic">Cucumber mosaic cucumovirus </Emphasis>Infection in Cowpea

We previously demonstrated that cowpea [Vigna unguiculata (L.) Walp.] cultivar Kurodane-Sanjaku contains the Cry gene, which confers resistance against Cucumber mosaic cucumovirus infection. In this paper, randomly amplified polymorphic DNA (RAPD) analysis was carried out to tag the Cry locus. Bulked segregant analysis for RAPD resulted in many polymorphisms in amplified DNA patterns. Candidates were further screened using parental and/or F₂ cowpea DNAs. As a result, we obtained three RAPD markers, D13/E14-350, WA3-850 and OPE3-500, flanking the Cry locus. In addition, we amplified cowpea sequences coding for the putative nucleotide-binding site (NBS). Degenerate primers based on NBS sequences of tobacco N and Arabidopsis RPS2 disease resistance genes were used for polymerase chain reaction, and resultant products were cloned and sequenced. Among eight independent clones, cowpea resistance gene analog (CRGA) 5 showed a distinct polymorphism when used as a probe for restriction fragment length polymorphism analysis against the susceptible cowpea cultivar PI 189375 and a near-isogenic line for the Cry. Linkage analyses of these molecular markers showed that genetic distances of CRGA5, D13/E14-350, WA3-850 and OPE3-500 to the Cry locus were 0.7, 5.2, 11.5 and 24.5 cM, respectively. Received 16 December 1999/ Accepted in revised form 22 March 2000     

Nandina mosaic virus is an isolate of <Emphasis Type="Italic">Plantago asiatica mosaic virus</Emphasis>

The complete nucleotide sequence of the genome of Nandina mosaic virus (NaMV), which has tentatively been assigned to the genus Potexvirus, is reported. The sequence is 6066 nt in length, excluding the poly(A) tail, and contains ORFs coding for proteins of 155, 25, 12, 13, and 21 kDa (ORFs 1–4 and the CP), respectively. The genomic organization of the virus and the signature motifs in the putative protein products are similar to the data reported for potexviruses for which complete sequences are known. Phylogenetic comparisons indicated that NaMV is most closely related to Plantago asiatica mosaic virus (PlAMV). Pairwise comparisons of the sequence data for these two viruses indicate that, based on criteria recently proposed for genera within the family Flexiviridae, NaMV and PlAMV should be considered to be strains/isolates of the same viral species. Both NaMV and PlAMV were first reported in 1976 but, as PlAMV was sequenced first, this name should take precedence with the name NaMV being relegated to a synonym.     

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.     

Leaf Smut of <Emphasis Type="Italic">Sagittaria latifolia </Emphasis>Caused by <Emphasis Type="Italic">Doassansia horiana</Emphasis>

A smut-like disease was found on the leaves of Sagittaria latifolia in Japan. Spore balls collected from the leaves of S. latifolia and S. trifolia var. edulis were used to cross-inoculate leaves of pathogen-free plants of the two species to identify the pathogen. Spots and swellings formed on leaves of the two species 10 days after inoculation. These symptoms were quite similar to those of the leaf smut disease of S. trifolia var. edulis caused by Doassansia horiana, and the spore balls were characteristic of the fungus. Therefore, the authors conclude that D. horiana caused leaf smut disease on S. latifolia. Received 18 January 2000/ Accepted in revised form 14 May 2000