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.     

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.     

Characterization of a novel potyvirus tentatively named <Emphasis Type="Italic">Ornithogalum</Emphasis> virus 3, successfully isolated from <Emphasis Type="Italic">O. thyrsoides</Emphasis> co-infected with two other potyviruses by single-aphid inoculation

A potyvirus tentatively named Ornithogalum virus 3 (OV-3) was successfully isolated by single-aphid transmissions from O. thyrsoides mix-infected with OV-3, Ornithogalum mosaic virus (OrMV) and Ornithogalum stripe mosaic virus (OrSMV). OV-3, a flexuous, rod-shaped particle of ca. 690 nm, was sap and aphid transmissible. The virus had a narrow host range and caused necrotic mosaic on O. thyrsoides under cold conditions. We therefore propose the name Ornithogalum necrotic mosaic virus (OrNMV) for OV-3. A synergistic increase in symptom severity was apparent on O. thyrsoides mix-infected with OrSMV/OrNMV, but not with either OrMV/OrNMV or OrMV/OrSMV. The nucleotide sequence data reported is available in the DDBJ/EMBL/GenBank databases under accession number AB282754.     

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.     

The <Emphasis Type="Italic">cyv-2</Emphasis> resistance to <Emphasis Type="Italic">Clover yellow vein virus</Emphasis> in pea is controlled by the eukaryotic initiation factor 4E

The same mutant allele of eukaryotic initiation factor 4E (eIF4E) that confers resistance to Pea seed-borne mosaic virus (sbm-1) and the white lupine strain of Bean yellow mosaic virus (wlv) also confers resistance to Clover yellow vein virus (ClYVV) in pea. The eIF4E genes from several pea lines were isolated and sequenced. Analysis of the eIF4E amino acid sequences from several resistant lines revealed that some lines, including PI 378159, have the same sequence as reported for sbm-1 and wlv. When eIF4E from a susceptible pea line was expressed from a ClYVV vector after mechanical inoculation of resistant PI 378159, the virus caused systemic infection, similar to its effects in susceptible line PI 250438. The resistance to ClYVV in line PI 378159 was characterized through a cross with PI 193835, which reportedly carries cyv-2. Mechanical inoculation of the F1 progeny with ClYVV resulted in no infection, indicating that the resistance gene in PI 378159 is identical to cyv-2 in PI 193835. Furthermore, particle bombardment of pea line PI 193835 with infectious cDNA of ClYVV (pClYVV/C3-S65T) resulted in the same resistance mode as that described for PI 378159. These results demonstrate that the resistance to ClYVV conferred by cyv-2 is mediated by eIF4E and that cyv-2 is identical to sbm-1 and wlv.     

Defective Long-distance Transport of <Emphasis Type="Italic">Cucumber mosaic virus </Emphasis>in Radish Is Efficiently Complemented by <Emphasis Type="Italic">Turnip mosaic virus</Emphasis>

A strain of Cucumber mosaic virus (CMV-D8) systemically infects Japanese radish (Raphanus sativus), but the Y strain of CMV (CMV-Y) only infects the inoculated leaves. Both of these strains cause severe systemic mosaic on the plants after dual infection with Turnip mosaic virus (TuMV). Synergistic interactions on long-distance transport of CMV-Y and CMV-D8 with TuMV were analyzed using an immunobinding assay. Direct tissue blots probed with either anti-CMV-Y or anti-TuMV antiserum clearly showed that CMV-Y efficiently spread and accumulated in the tissues of noninoculated upper leaves and roots when co-inoculated with TuMV, and that long-distance movement of CMV-D8 was enhanced by the presence of TuMV. Received 16 September 1999/ Accepted in revised form 5 February 2000     

Molecular identification of a new isolate of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup II from basil (<Emphasis Type="Italic">Ocimum sanctum</Emphasis>) in India

Natural occurrence of mosaic disease was observed on basil (Ocimum sanctum L.) in Aligarh, U. P., India, during 2008. The disease could be transmitted by sap inoculations from naturally infected O. sanctum to O. sanctum and some test plant species. Cucumber mosaic virus (CMV) was detected by RT-PCR using coat protein gene specific primers of CMV (Acc. AM180922 & AM180923), which resulted in the expected size ~650 bp amplicon in infected samples. The amplicon was cloned, sequenced and data were deposited in GenBank Acc. EU600216. The sequence data analysis revealed 97–99% identities at both nucleotide and amino acid levels with the CMV strains of subgroup II reported worldwide. Based on the high sequence identities and close phylogenetic relationships with CMV subgroup II strains, the virus under study has been identified as a new isolate of CMV subgroup II and designated as CMV-Basil.     

<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.     

Multiple virus infections of lablab [<Emphasis Type="Italic">Lablab purpureus</Emphasis> (L.) Sweet] in Nigeria

Leaf samples of Lablab purpureus collected from two agroecological zones of Nigeria—the northern guinea savanna zone (NGSZ) and the derived savanna zone (DSZ)—were infected with viruses when serologically indexed against available antisera. Approximately 31.1 and 81.1% of the leaf samples collected from the NGSZ and DSZ, respectively, were infected. Seven viruses were found: Bean common mosaic virus (BCMV), Cowpea aphid-borne mosaic virus (CABMV), Cucumber mosaic virus (CMV), Cowpea mottle virus (CPMoV), Cowpea severe mosaic virus (CPSMV), Southern bean mosaic virus (SBMV) and Tobacco mosaic virus (TMV) were detected from samples collected from NGSZ, while CMV, CPMoV, Cowpea mosaic virus (CPMV) and CPSMV were detected from samples from DSZ.     

Molecular characterisation of <Emphasis Type="Italic">Rice stripe necrosis virus</Emphasis> as a new species of the genus <Emphasis Type="Italic">Benyvirus</Emphasis>

The complete nucleotide sequences of RNAs 1 and 2 of Rice stripe necrosis virus (RSNV) were determined and compared to the corresponding genomes of all sequenced, rod-shaped plant viruses. The genome organisation of RSNV RNA1 and RNA2 is nearly identical to that of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV), definitive species of the genus Benyvirus. As demonstrated for BNYVV and BSBMV, the RNA1 of RSNV also encodes a single ORF with putative replicase-associated motifs, which distinguishes benyviruses from all other viruses possessing rod-shaped particles. As described for BNYVV, RNSV RNA-2 also contains six ORFs: the capsid protein gene, the read-through protein gene, a triple gene block gene that codes for three different proteins, and a 17 kDa cysteine-rich protein. RNAs 3 and 4 (or 5 in the case of BNYVV), identified in natural infections of BNYVV and BSBMV, were not detected in any of the 44 RSNV cDNA clones obtained in this investigation. Nevertheless, phylogenetic and amino comparative acid sequence analyses demonstrated that RSNV is more closely related to BNYVV and BSBMV than to any other rod-shaped plant virus characterised to date.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

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">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

Fusarium rot of hyacinth caused by <Emphasis Type="Italic">Gibberella zeae</Emphasis> (anamorph: <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic"> graminearum</Emphasis>)

Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium rot of hyacinth” as a new disease because only the anamorph, F. graminearum, was identified on the diseased host plant. The authors contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively.     

<Emphasis Type="Italic">In vitro</Emphasis> Selection of Maize Rhizobacteria to Study Potential Biological Control of <Emphasis Type="Italic">Aspergillus</Emphasis> Section <Emphasis Type="Italic">Flavi</Emphasis> and Aflatoxin Production

The aims of this study were to select bacterial isolates from the non-rhizophere of maize soil and to examine their antagonistic activity against Aspergillus section Flavi strains. The first selection was made through ecophysiological responses of bacterial isolates to water activity (a_w) and temperature stress. Subsequently, an Index of Dominance test (I_D), ecological similarity and inhibition of the lag phase prior to growth, growth rate and aflatoxin B₁ accumulation were used as criteria. From the first assay nine bacterial strains were selected. They grew well at 25 and 30 °C, with growth optima between 0.982 and 0.955 a_W using 48 h of incubation. There was ecological similarity between the bacterial strains Bacillus subtilis (RCB 3, RCB 6), Pseudomonas solanacearum RCB 5, Amphibacillus xylanus RCB 27 and aflatoxigenic Aspergillus section Flavi strains at 0.982 at 25 °C. The predominant interaction between all selected bacteria and fungi in dual culture was mutual intermingling at 0.982. Mutual inhibition on contact and mutual inhibition at a distance was observed at 0.955 a_w, between only four bacteria and some Aspergillus strains. Bacillus subtilis RCB 55 showed antifungal activity against Aspergillus section Flavi strains. Amphibacillus xylanus RCB 27, B.␣subtilis RCB 90 and Sporolactobacillus inulinus RCB 196 increased the lag phase prior to growth and decreased the growth rate of Aspergillus section Flavi strains. Bacillus subtilis strains (RCB 6, RCB 55, RCB 90) and P. solanacearum RCB 110 inhibited aflatoxin accumulation. Bacillus subtilis RCB 90 completely inhibited aflatoxin B₁ accumulation at 0.982 a_W. These results show that the bacterial strains selected have potential for controlling Aspergillus section Flavi over a wide range of relevant environmental conditions in the stored maize ecosystem.     

Response of UK winter wheat cultivars to <Emphasis Type="Italic">Soil-borne cereal mosaic</Emphasis> and <Emphasis Type="Italic">Wheat spindle streak mosaic viruses</Emphasis> across Europe.

Twenty-one UK winter wheat cultivars were grown over three seasons at sites with natural inoculum sources of Soil-borne cereal mosaic virus (SBCMV) and Wheat spindle streak mosaic virus (WSSMV) located in France, Italy and the UK. Plants were assessed visually for virus symptoms and leaf extracts were tested for the presence of each virus using enzyme-linked immunosorbent assays (ELISA). Cultivars showing little or no foliar symptoms and low levels of virus in leaf tissue were classified as resistant to each virus. All the trials were taken to harvest and agronomic data collected. At the most heavily infected sites, severe symptoms of SBCMV were observed in all UK cultivars except Aardvark, Charger, Claire, Cockpit, Hereward and Xi 19. The latter cultivars exhibited either light or no symptoms and little or no SBCMV infection in leaves. In fields with WSSMV, the virus failed to develop in Italy, but was detected in the leaves of all the susceptible control cultivars at a site in France. However, no UK cultivar tested positive for WSSMV. Multi-site analysis indicated that the presence of WSSMV did not increase the impact of SBCMV on the height, thousand-grain weight or yield of UK cultivars. The wheat cultivars on test gave a similar response to SBCMV across three European countries. Possible sources of SBCMV resistance are discussed.