Molecular characterization and detection of European isolates of Soil-borne wheat mosaic virus

Sequencing of a recently identified isolate of Soil-borne wheat mosaic virus (SBWMV) from the UK confirmed its identity as a European strain of the species and provided further evidence for taxonomic divisions in the group. Two RT–PCR protocols were developed for the detection of all SBWMV strains and for the specific detection of the European SBWMV strain, and were tested successfully on 21 isolates of SBWMV from a range of countries. Both protocols worked well using either purified total RNA in one- or two-step RT-PCR, or immunocapture (IC) RT–PCR. The sensitivity of IC RT-PCR was 100 times greater than ELISA. Neither set of primers produced any PCR product with either Wheat spindle streak mosaic virus or Wheat yellow mosaic virus which are frequently associated with SBWMV, or with the related viruses Indian peanut clump virus , Potato mop-top virus , Beet soil-borne virus and Beet necrotic yellow vein virus . This new diagnostic protocol will improve disease management by enabling correct identification of the causal pathogen and earlier detection than is possible serologically.     

A novel putative member of the family Benyviridae is associated with soilborne wheat mosaic disease in Brazil

Soilborne wheat mosaic disease (SBWMD), originally attributed to infections by Soilborne wheat mosaic virus (SBWMV) and Wheat spindle streak mosaic virus (WSSMV), is one of the most frequent virus diseases and causes economic losses in wheat in southern Brazil. This study aimed to characterize molecularly the viral species associated with wheat plants showing mosaic symptoms in Brazil. Wheat leaves and stems displaying mosaic symptoms were collected from different wheat cultivars in Passo Fundo municipality, Rio Grande do Sul State, southern Brazil. Double-stranded RNA was extracted and submitted to cDNA library synthesis and next-generation sequencing. No sequences of SBWMV and WSSMV were detected but the complete genome sequence of a putative new member of the family Benyviridae was determined, for which the name wheat stripe mosaic virus (WhSMV) is proposed. WhSMV has a bipartite genome with RNA 1 and RNA 2 organization similar to that of viruses belonging to Benyviridae. WhSMV RNA 1 has a single open reading frame (ORF) encoding a polyprotein with putative viral replicase function. WhSMV RNA 2 has six ORFs encoding the coat protein, the major protein (read-through), triple gene block movement proteins (TGB 1, 2 and 3) and ORF 6 (hypothetical protein). In addition to the genomic organization and nucleotide and amino acid sequence identities, phylogenetic analyses also corroborated that WhSMV is a virus species of the Benyviridae. However, isolates of WhSMV formed a clade distinct from members of the genus Benyvirus. It was also demonstrated that the plasmodiophorid Polymyxa graminis is associated with wheat roots showing SBWMD symptoms and infected by WhSMV.     

Occurrence of viruses in irrigated wheat in Zambia

Surveys were conducted during the cool-dry months of June–August 1997 and June–July 1998 for the presence of viruses in irrigated wheat in Central, Copperbelt, Lusaka and Southern Provinces of Zambia in 14 commercial farms and four wheat cultivar plots. Virus symptoms were observed on nine wheat cultivars ( Triticum aestivum 'Deka', 'Gamtoos', 'Lorie II', 'MM2', 'Nata', 'Nkwazi', 'P7', 'Scan' and 'Sceptre') of South African, Zambian and Zimbabwean origin. Several viruses were identified on the basis of field symptomatology, symptoms developing on mechanically inoculated indicator plant species or cultivars and serology (DAS-ELISA). The study revealed the occurrence of Brome mosaic virus (BMV), Barley stripe mosaic virus (BSMV), Barley yellow dwarf virus and its strains (BYDV-PAV and RPV), Soil-borne wheat mosaic virus (SBWMV), Wheat dwarf virus (WDV), Wheat streak mosaic virus (WSMV) and Wheat spindle streak mosaic virus (WSSMV). DSA-ELISA tests confirmed these identifications. The prevalence of viruses varied annually and from field to field. BSMV, BYDV-PAV, SBWMV, WDV, WSMV and WSSMV were found to be the most prevalent viruses. Viruses generally occurred in mixed infections of 3–6 viruses and the most common virus complex consisted of 4 viruses (50%), viz. BYDV, SBWMV, WDV and WSSMV. Five- and six-virus complexes were relatively less common (20% each) whereas 3-virus complex was noticed in only 10% cases. SBWMV and WSSMV have been found to be new to Africa and Zambia and are reportedly vectored by a fungal protist – Polymyxa graminis . BYDV strains MAV and SGV were also tested but gave negative results against their antisera.     

Biology of a new virus isolated from Lupinus nootkatensis plants in Alaska

A new virus named Nootka lupine vein-clearing virus (NLVCV) was isolated from Lupinus nootkatensis plants that were confined to a relatively small area in the Talkeetna mountains of south-central Alaska. Annual surveys (2000–03) consistently found leaf symptoms of pronounced vein clearing and mosaic on 3- to 4-week-old plants in late June. Spherical particles ≈30 nm in diameter were isolated from these leaves. Virions contained a single-stranded RNA of ≈4·0–4·2 kb and one species of capsid protein estimated to be ≈40 kDa. The double-stranded RNA profile from naturally infected leaves consisted of three major bands ≈4·2, 1·9 and 1·5 kbp. Protein extractions from either sap or virions of diseased plants reacted to polyclonal antiserum made against the virions in Western blot assays. A predicted PCR product ≈500 bp was synthesized from virion RNA using primers specific to the carmovirus RNA-dependent RNA polymerase (RDRP) gene. The nucleotide sequence of the amplified DNA did not match any known virus, but contained short regions of identity to several carmoviruses. Only species belonging to the Fabaceae were susceptible to NLVCV by mechanical inoculation. Based on dsRNA profile, size of virion RNA genome and capsid protein, and similarity of the RDRP gene to that of other carmoviruses, it is suggested that NLVCV is a member of the family Tombusviridae , and tentatively of the genus Carmovirus . As the host range, RDRP gene and dsRNA profile of NLVCV are different from those of known viruses, this is a newly described plant virus.     

Monoclonal antibodies detect a single amino Acid difference between the coat proteins of soilborne wheat mosaic virus isolates: implications for virus structure

ABSTRACT Four monoclonal antibodies (MAbs) were prepared against an isolate of soilborne wheat mosaic furovirus from Oklahoma (SBWMV Okl-7). Three MAbs had different reactivities in tests on SBWMV isolates from Nebraska (Lab1), France, and Japan. One MAb (SCR 133) also reacted with oat golden stripe furovirus. None of the MAbs cross-reacted with other rod-shaped viruses including beet necrotic yellow vein furovirus, potato mop-top furovirus, and tobacco rattle tobravirus. Sequence analysis of nucleotides between 334 and 1,000 of RNA 2, the region that encodes the coat protein (CP) and the first 44 amino acids of a readthrough protein, of the four SBWMV isolates revealed up to 27 base changes from the published sequence of a Nebraska field isolate of SBWMV. Most changes were translationally silent, but some caused differences of one to three amino acids in residues located near either the N- or C-terminus of the CPs of the different isolates. Two further single amino acid changes were found at the beginning of the readthrough domain of the CP-readthrough protein. Some of these amino acid changes could be discriminated by MAbs SCR 132, SCR 133, and SCR 134. Peptide scanning (Pepscan) analysis indicated that the epitope recognized by SCR 134 is located near the N-terminus of the CP. SCR 132 was deduced to react with a discontinuous CP epitope near the C-terminus, and SCR 133 reacted with a surface-located continuous epitope also near the C-terminus. Predictions of CP structure from computer-assisted three-dimensional model building, by comparison with the X-ray fiber diffraction structure of tobacco mosaic virus, suggested that the three CP amino acids found to differ between isolates of SBWMV were located near the viral surface and were in regions predicted to be antigenic.     

Identification and characterization of peanut stunt cucumovirus from naturally infected alfalfa in Iran

A mechanically transmissible virus was isolated from a naturally infected alfalfa plant ( Medicago sativa ) in Karaj, Iran. It induced fern-leaf symptoms in Lycopersicon esculentum , general chlorosis and stunting in Arachis hypogoea , local lesions and systemic infection in Chenopodium quinoa , Phaseolus vulgaris and Vigna unguiculata but only local lesions in C. amaranticolor . In gel-immunodiffusion tests, it reacted strongly with an antiserum to peanut stunt cucumovirus (PSV), moderately with two of six antisera to cucumber mosaic cucumovirus (CMV) and with two of five antisera to tomato aspermy cucumovirus. Its spherical virions (28 nm in diameter) contained a coat protein of approximately 29 kDa and encapsidated four species of RNAs with similar electrophoretic mobilities to RNAs 1–4 of CMV strains Fny and LS and those of PSV strains J and W. Its encapsidated RNAs hybridized in slot-blot hybridization assay with the complementary DNA probe of PSV-W RNAs but not with those of CMV strains. Therefore, on the basis of biological, serological and physico-biochemical properties, the virus was identified as PSV. No satellite RNA was associated with the virus. This is the first report of PSV in Iran.     

Biological, serological and molecular characterization of a cucumber mosaic virus isolate from India

A. virus causing mosaic and leaf deformation of Physalis minima has been identified as an isolate of cucumber mosaic virus (CMV) on the basis of its transmission by aphids in a non-persistent manner, polyhedral particles of 29 nm diameter, molecular weight of coat protein subunits us 24-5 kDa. serological relationship with a CMV isolate and a tripartite single-stranded RNA genome with a subgenomic RNA4- Furthermore. cDNA representing coat protein gene was synthesized and cloned. Complete nucleotide sequences (890 nt) were obtained which showed a coat protein gene open reading frame of 657 residues. THE nucleotide sequences provided the 218 amino ACID sequences of the coat protein. Nucleotide as well as amino acid sequences revealed more than 90% identity with the CMV subgroup I strains.     

感染土传小麦花叶病毒的小麦花叶病的电子显微镜研究

 经对山东省崂山地区获得的自然感病的小麦病叶进行了电镜的观察和研究。指出在早春时节小麦返青以后很快开始显示症状,沿叶脉出现短的褪绿色条纹,植株生长受阻,矮缩,黄化以至不能抽穗。该病毒粒子为细长的杆状,长度120~270nm,直径为18~20nm。值得注意的是在狭窄的胞质区可经常见到聚集的病毒颗粒,其中大部分病毒附着在膜结构上。当横切时病毒颗粒呈电子透明的空心,围绕这个空心周围是一个着色很深的直径约为10nm的核酸环,围绕这个核酸环周围是一个密度不大的蛋白外壳。病毒颗粒的聚集大多与很多弯曲的管状物的形成聚集相伴随着。也看到了完全由病毒颗粒所组成的晶体状聚集物。根据以上所述,该病原鉴定为土传小麦花叶病毒。     

Unterschiedliche Formen der Resistenz gegen bodenbürtige Viren des Weizens

In Germany the furovirus Soil-borne cereal mosaic virus (SBCMV) and the bymovirus Wheat spindle streak mosaic virus (WSSMV) occur often together particularly in several rye production areas. Soil-borne wheat mosaic virus (SBWMV), a wheat infecting furovirus, has so far been found only in one field near Heidelberg. Each of these viruses is transmitted by Polymyxa graminis. The cultivation of resistant varieties is the only promising measure to prevent yield losses caused by soil-borne viruses. Resistance of wheat against the bymovirus WSSMV is comparable to the immunity of barley to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In case of immunity no virus multiplication is observed in resistant cultivars. In contrast, all wheat cultivars are hosts of the furoviruses. All cultivars – including the resistant ones – can be infected following mechanical inoculation with SBWMV and SBCMV. Resistance to furoviruses is based on reduced levels of virus multiplication in roots and on inhibition of virus movement from roots to leaves. Because of the inhibited virus movement from roots to aerial parts of plants this type of resistance is referred to as translocation resistance. In spite of the different resistance mechanisms the absence of virus symptoms on the leaves is a common selection criterion for both immunity and translocation resistance. Therefore, the symptom free development of plants on uniformly infested fields is the best criterion for selecting wheat lines with resistance to soil-borne viruses. The limited suitability of other selection methods is discussed.     

Pfaffia mosaic virus: a new potyvirus found infecting Pfaffia glomerata in Brazil

In Brazil plants of Pfaffia glomerata with mosaic symptoms were found to be infected with a previously undescribed potyvirus, Pfaffia mosaic virus (PfMV). Virus particles were long and flexuous, c.  10 × 700–800 nm, and cylindrical inclusions typical of potyviruses were present in cells of infected tissue. Partial host-range studies revealed that in addition to P. glomerata , PfMV infected only Chenopodium amaranticolor and Chenopodium quinoa . It was efficiently transmitted by the aphids Aphis gossypii and Myzus persicae . Polyclonal antiserum produced against the PfMV coat protein (CP) reacted with Potato virus Y (PVY), but not with four other potyviruses in PTA-ELISA. The similarity of the nucleotide sequence of the PfMV coat-protein gene ( CP ) varied from 7 to 76% when compared with other members of the family Potyviridae . Similarity of the 3' NTR sequence varied from 4 to 23%. In both cases the highest similarity was with PVY. These data indicate that PfMV is a new species in the genus Potyvirus .     

Untersuchungen zum Infektionsverlauf und zur biologischen Differenzierung von bodenbürtigen Viren in Roggen, Triticale und Weizen

Abstract In the frame of the investigation of epidemiology of soil-borne viruses, like the Soil-borne cereal mosaic virus (SBCMV), Soil-borne wheat mosaic virus (SBWMV) and the Bymovirus Wheat spindle streak mosaic virus (WSSMV), which were transmitted by fungal vector Polymyxa graminis Ledingham, the infection progress in different cereals was observed. The detection of furovirus and bymovirus in field plants was depending on temperature conditions during the vegetation period and the kind of cereals. The furoviruses tolerate a broad temperature spectrum and once established infection is detectable until the harvest time. In contrast to this observation, the propagation of WSSMV seems to be restricted to lower temperatures. Consequently, this virus is detected best at the end of February until the middle of April. Among the tested cereals, rye becomes more early infected than wheat and triticale. Both furoviruses could be differed by variable virulence reactions on cereal hosts and indicator plants. The SBCMV infects rye, triticale and wheat but not barley. The SBWMV is able to contaminate beside these cultures barley too. Both viruses are distinguished in the infection typ in Nicotiana benthamiana. Whereas SBCMV isolates spread out in the whole plant and cause yellowing and the die back of plants, the SBWMV infects the inoculated leaves only.     

Natural Infection of Wheat by the Type Strain of Soil-borne Wheat Mosaic Virus in a Field in Southern Germany

Molecular analyses revealed that a virus causing a severe disease of wheat in one field in Southern Germany is closely related to the Nebraska type strain of Soil-borne wheat mosaic virus (SBWMV) and only distantly related to Soil-borne cereal mosaic virus that is widely distributed in Europe. The latter virus was not found in the SBWMV-containing leaf samples. This is the first report of the occurrence of SBWMV in Germany, and perhaps in all of Europe, which has been confirmed on the molecular level.     

Identification of potyviruses infecting vanilla by direct sequencing of a short RT-PCR amplicon

A simple one-tube one-step RT-PCR assay with degenerate primers followed by direct sequencing of a 327 bp coat protein gene fragment was used to identify the potyviruses infecting vanilla. With this technique, unambiguous species allocation was achieved for 34 potyvirus-infected vanilla samples collected in the Indian Ocean and the Pacific areas between 1997 and 2005. Virus identification relied on blast homology and nucleotide identities of 162 to 327 nt fragments with known potyvirus sequences. Species allocation was confirmed by neighbour-joining of the 149 nt common to 32 vanilla sequences and 51 known potyviruses. Subject to further identification, these data revealed four additional Potyvirus species that may infect vanilla: Bean yellow mosaic virus , Cowpea aphid-borne mosaic virus , Ornithogalum mosaic virus and Wisteria vein mosaic virus . The procedure was rapid, cost-effective, easy to use and showed a good taxonomic discriminating value. It also enabled the identification of potyviruses in adjacent weeds and should thus aid the understanding of outbreaks of potyviruses occurring in varied epidemiological circumstances.     

发生在我国的小麦黄花叶病毒病

本文对山东荣成流行的一种小麦病毒病进行了鉴定。提纯的病毒颗粒为长线状,13×100—300nm及13×350—650nm。汁液接种感染小麦,但不感染烟草、苋色藜等植物。病土、病根以及含有禾谷类多粘菌(POlymyxa graminis)游动孢子的浸液可以传毒于小麦、大麦及黑麦。此病毒与大麦黄花叶病毒(BYMV)、小麦梭条花叶病毒(WSSMV)有血清学关系,与小麦土传花叶病毒(WSBMV)无血清学关系。病叶表皮细胞中有无定形内含体。超薄切片可见风轮状内含体。实验结果表明,荣城地区发生的这种小麦病毒病是小麦黄花叶病毒所致。     

A new furovirus infecting barley in France closely related to the Japanese soil-borne wheat mosaic virus

In April 2001, stunted barley plants bearing mosaic symptoms were observed in a field in France (Marne Department, 51). Rod-shaped and flexuous particles were visualized by electron microscopy and positive serological reactions were detected by ELISA with Barley yellow mosaic virus (BaYMV) and Soil-borne cereal mosaic virus (SBCMV) polyclonal antisera. The tubular virus which was soil transmissible to barley cv. Esterel was separated from BaYMV by serial mechanical inoculations to barley cv. Esterel. This furo-like virus, in contrast to a French isolate of SBCMV, could be transmitted to Hordeum vulgare, Avena sativa, Beta vulgaris and Datura stramonium. RT-PCR was used to amplify the 3′-terminal 1500 nucleotides of RNA1 and the almost complete sequence of RNA2. Nucleotide and amino acid sequence analyses revealed that the French virus infecting barley is closely related to a Japanese isolate of Soil-borne wheat mosaic virus (SBWMV-JT) which was originally isolated from barley. This French isolate was named SBWMV-Mar. The 3′ UTRs of both RNAs can be folded into tRNA-like structures which are preceded by a predicted upstream pseudoknot domain with seven and four pseudoknots for RNA1 and RNA2, respectively. The four pseudoknots strongly conserved in RNAs 1 and 2 of SBWMV-Mar show strong similarities to those described earlier in SBWMV RNA2 and were also found in the 3′ UTR of Oat golden stripe virus RNAs 1 and 2 and Chinese wheat mosaic virus RNA2. Sequence analyses revealed that the RNAs 2 of SBWMV-Mar and -JT are likely to be the product of a recombination event between the 3′ UTRs of the RNAs 2 of SBWMV and SBCMV. This is the first report of the occurrence of an isolate closely related to SBWMV-JT outside of Japan.     

烟草坏死病毒A大豆分离物的分子鉴定

 对曹琦和濮祖芹早期分离到的烟草坏死病毒大豆分离物的生物学、血清学和外壳蛋白的序列进行了进一步研究。该分离物能侵染8科29种植物, 除系统侵染大豆和本生烟外, 其余寄主均为局部侵染。电镜下病毒粒子呈球状, 直径约28nm。基因组为单组分RNA, 大小约为3.7 kb, 具有2条亚基因组, 分别约为1.6 kb和1.3 kb。外壳蛋白亚基的分子量约为30 kDa。血清学试验表明, 该分离物与TNV柳树分离物的抗血清呈特异反应, 与同属坏死病毒属(Necrovirus)的烟草坏死病毒D(TNV-D)和甜菜黑色焦枯病毒(BBSV)无血清学关系。利用简并引物通过RT-PCR克隆了该分离物的外壳蛋白基因。序列分析表明, 该分离物与烟草坏死病毒A(TNV-A)、TNV-D和TNV-D^H的外壳蛋白分别具有88.77%、45.13%和45.49%的氨基酸序列一致性。因此, 该大豆分离物属于TNV-A的一个新株系, 命名为TNV-A^C。     

Response of Hard Red Winter Wheat to Soilborne wheat mosaic virus Using Novel Inoculation Methods

ABSTRACT Soilborne wheat mosaic virus (SBWMV) is an agronomically important pathogen of wheat that is transmitted by the soilborne plasmodiophorid vector Polymyxa graminis. In the laboratory, attempts to generate SBWMV-infected plants are often hampered by poor infectivity of the virus. To analyze the mechanism for virus resistance in wheat cultivars, we developed novel inoculation techniques. A new technique for foliar inoculation of SBWMV was developed that eliminated wound-induced necrosis normally associated with rub inoculating virus to wheat leaves. This new technique is important because we can now uniformly inoculate plants in the laboratory for studies of host resistance mechanisms in the inoculated leaf. Additionally, wheat plants were grown hydroponically in seed germination pouches and their roots were inoculated with SBWMV either by placing P. graminis-infested root material in the pouch or by mechanically inoculating the roots with purified virus. The susceptibility of one SBWMV susceptible and three field resistant wheat cultivars were analyzed following inoculation of plants using these novel inoculation techniques or the conventional inoculation technique of growing plants in P. graminis-infested soil. The results presented in this study suggest that virus resistance in wheat likely functions in the roots to block virus infection.     

The effects of postplanting environment on the incidence of soilborne viral diseases in winter cereals

ABSTRACT Soilborne wheat mosaic virus (SBWMV) and Wheat spindle streak mosaic virus (WSSMV) are putatively transmitted to small grains by the obligate parasite Polymyxa graminis, but little is known about environmental requirements for transmission and the resulting disease incidence. We planted susceptible wheat and triticale cultivars in field nurseries on different autumn dates in 3 years and observed the incidence of symptomatic plants in each following spring. Autumn postplanting environment explained most of the variation in disease caused by both viruses. Little apparent transmission, based on eventual symptom development, of either virus occurred after the average soil temperature dropped below 7 degrees C for the remainder of the winter. To forecast disease, we tested an SBWMV transmission model in the field, based on laboratory results, that predicts opportunities for transmission based on soil temperature and soil moisture being simultaneously conducive. This model was predictive of soilborne wheat mosaic in 2 of 3 years. Zoospores of P. graminis have optimal activity at temperatures similar to those in the SBWMV transmission model. Furthermore, the matric potential threshold (as it relates to waterfilled pore sizes) in the SBWMV transmission model fits well with P. graminis as vector given the size restrictions of P. graminis zoospores. Conditions optimal for SBWMV transmission in the laboratory were not conducive for WSSMV transmission in the laboratory or for wheat spindle streak mosaic development in the field. This differential response to environment after emergence, as indicated by disease symptoms, may be due to virus-specific environmental conditions required to establish systemic infection via the same vector. Alternatively, the differential response may have been due to the involvement of a different vector in our WSSMV nursery than in our SBWMV nursery. Our results suggest that, as a control tactic for SBWMV or WSSMV, earliness or lateness of planting is less important in determining virus transmission and disease than the specific postplanting environment. Improved models based on the postplanting environment might predict virus-induced losses of yield potential, and in some cases, growers might avoid purchase of spring inputs such as pesticides and fertilizer for fields with greatly reduced yield potential.