Characteristics for Practical Use of Attenuated Isolate UA-Fukushima of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis>

L₁₁A-Fukushima (L₁₁A-F) derived from attenuated isolate LuA of Tomato mosaic virus (ToMV) has the highest ability to cross protect against virulent ToMV among LuA and its derivatives and is stably inherited. Growth, yield, fruit quality and symptom attenuation of inoculated tomato plants did not differ significantly between L₁₁A-F and L₁₁A. The infectivity of progeny viruses in tomato infected with LuA-F was less than 4% of that with virulent ToMV. From these results, L₁₁A-F appears to possess the properties necessary for practical use. To manage L₁₁A-F strictly, a PCR-based assay to detect trace contamination of virulent ToMV in L₁₁A-F preparations was established. Received 10 June 2002/ Accepted in revised form 30 October 2002     

黄瓜花叶病毒外壳蛋白质进入叶绿体与症状发生的关系

 由感染黄瓜花叶病毒(CMV)、健康和转外壳蛋白(CP)基因的烟叶以原生质体法制备纯化的叶绿体。经SDS-PAGE电泳,银染色,比较其蛋白质图谱。用CMV外壳蛋白游离亚基制备的抗血清进行Western blotting。结果发现:1.CP存在于CMV侵染的烟叶绿体中。2.叶绿体中CP的浓度和花叶症状严重程度呈正相关;3.表达CP的转基因烟草叶绿体中未测出CP存在。根据以上结果,认为CMV侵染的烟花叶症状产生与CP进入叶绿体有直接相关性。     

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.     

Kazakh strains of tobacco mosaic virus: two strains with potentially destabilizing amino acid substitutions in the coat protein

Some properties of two Kazakh strains (K1 and K2) of tobacco mosaic virus (TMV) are described. K1 had been isolated by Dr M. Gol'din in 1963, and K2 recently in our laboratory. Both strains were rather similar in host range and antigenic properties to the tomato strain of TMV (tomato mosaic virus, ToMV), but differed from the latter by inducing unusual symptoms on upper noninoculated leaves of infected tobacco plants. K1 was semi-defective and temperature-sensitive, and formed large amounts of long RNA-free helical protein rods in infected plants. K2 was found to be neither defective nor temperature-sensitive, and did not produce protein rods in infected cells. K1 and K2 coat protein gene sequencing data showed, as expected, that both proteins are similar in primary structure to ToMV coat protein: only three amino acid substitutions, relative to ToMV, were found in K1 and five in K2 coat protein. Two of these substitutions are unusual, namely, substitution of normally strictly conserved R92 by S (with concomitant Q99→R change) in K2 and substitution of K53 by E in K1.     

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.     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.     

Discrimination between Virulent and Attenuated Isolates of Tomato mosaic virus by Restriction Fragment Length Polymorphism

The three missense mutations on the gene for the 130-K protein of Tomato mosaic virus (ToMV) L₁₁A have been thought to be responsible for the attenuation of its virulence. The Eco47I RFLP detecting the missense mutation at 2349 successfully discriminated L₁₁A and its derivative attenuated isolates from ToMV virulent ones. RFLP analysis and mismatch amplification assay detecting the missense mutations at 1117 and 2754, respectively, could not discriminate some of the attenuated isolates from the virulent ones. These results indicated that, of the three missense mutations, only the one at 2349 was conserved in all the L₁₁A-derivative attenuated isolates. Received 16 March 2001/ Accepted in revised form 22 June 2001     

Viruses of pepper in plastic houses in Crete

During a survey of virus diseases affecting pepper grown in plastic houses in Crete, during 1984–1986, tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) were detected. The most common virus was TMV, being present in samples of alle pepper cultivars carrying the L₁ resistance gene, while ToMV was isolated only from susceptible pepper cultivars. According to responses ofCapsicum spp. the isolates from 640 samples checked were classified into three pathotypes: P₀, P_1.2 and P_1.2.3. Results of this study show that P_1.2 represents at present the major threat to the Cretan pepper industry.Samenvatting Bij een in de jaren 1984–1986 gehouden inventarisatie van virusziekten in paprika in plastic-foliekassen op Kreta werd zowel het tabaksmozaïekvirus (TMV) als het tomatemozaïekvirus (ToMV) aangetoond. TMV kwam zeer algemeen voor: het werd aangetroffen in alle monsters van de paprikarassen met het resistentiegen L₁. ToMV werd alleen geïsoleerd uit planten van vatbare paprikarassen. Volgens de waargenomen symptomen die de isolaten van 640 monsters opCapsicum spp. vertoonden, konden de isolaten in drie pathotypen, nl. P₁, P_1,2 en P_1,2,3, worden geklassificeerd. Uit het onderzoek is gebleken dat P_1,2 de belangrijkste bedreiging vormt voor de teelt van paprika op Kreta.     

Molecular Characterization of <Emphasis Type="Italic">Bean yellow mosaic virus </Emphasis>from Vegetatively Propagated Dwarf <Emphasis Type="Italic">Gentiana </Emphasis>Plants

The causative virus (isolate No. 4) of gentian (Gentiana spp.) mosaic, which had been identified previously as Clover yellow vein virus (C1YVV) on the basis of host range and serological reactions, was re-identified as Bean yellow mosaic virus (BYMV) on the basis of the nucleotide sequences of the gene for the coat protein (CP) and the 3′-noncoding region, as well as the predicted amino acid sequence of CP. Received 16 April 2002/ Accepted in revised form 19 June 2002     

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.     

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">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.     

Genetic variability and population structure of <Emphasis Type="Italic">Grapevine virus A</Emphasis> coat protein gene from naturally infected Italian vines

Grapevine virus A (GVA) is considered one of the viruses associated with rugose wood (RW), one of the most economically important diseases of grapevine. Thirty-seven GVA isolates collected from grapevine cultivars from Marche (central-eastern Italy), Apulia and Campania (southern Italy), were subjected to molecular characterization. The genetic and population diversity was studied in the coat protein (CP) gene by RT-PCR-RFLP analysis with three restriction enzymes (MseI, AluI, and AciI), and nucleotide sequencing. A new primer pair (CP1F/R) allowing amplification of the whole CP gene (621 bp) was developed. RFLP with AciI yielded the highest number of variants in GVA isolates, showing seven different ‘simple’ profiles (A, B, C, D, E, F, and G). ‘Complex’ profiles were also found, and the most common variant combination was A + B in 39% of isolates. The analysis of GVA sequences confirmed the presence of plants infected with more than one GVA variant and suggested that RT-PCR-RFLP is suitable for evaluating population diversity of GVA enabling a screening of different haplotypes. The distribution of RFLP profiles and the phylogenetic analysis were not correlated with the location of infected plants, showing the presence of a GVA population with genetic diversity in the average with those of RNA viruses.     

Characterization of symptom determinants in two mutants of cucumber mosaic virus Y strain,causing distinct mild green mosaic symptoms in tobacco

Two mutants of Cucumber mosaic virus, CMV(Y/GM1) and CMV(Y/GM2), which induced mild green mosaic symptoms in tobacco, were isolated from plants regenerated from tobacco leaves with yellow mosaic symptoms originally infected with the yellow strain of CMV [CMV(Y)]. Although the appearance of mild green mosaic symptoms in tobacco infected with CMV(Y/GM2) was unstable, CMV(Y/GM3) derived from CMV(Y/GM2) reproducibly induced mild green mosaic symptoms in tobacco similar to CMV(Y/GM1). A comparison of the deduced amino acid sequences of the coat proteins of CMV(Y), CMV(Y/GM1) and CMV(Y/GM3), showed single amino acid substitutions from Thr to Ile at position 124 in the CMV(Y/GM1) coat protein and from Val to Ile at position 111 in the CMV(Y/GM3) coat protein. When the amino acid at the 124 or 111 position in the CMV(Y) coat protein was changed to Ile at the cDNA level, CMV RNA3 transcribed in vitro from each cDNA induced mild green mosaic symptoms in tobacco after inoculation with in vitro transcribed CMV(Y) RNA1 and RNA2. The results indicated that amino acids at positions 111 and 124 in the coat protein were responsible for the phenotypic changes caused by the two CMV isolates.     

Indexing system for<Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) in commercial tomato seed lots

An indexing system for detectingTomato mosaic virus (ToMV) in commercial tomato seed lots is described. Factors associated with the procedure were analyzed and the following standard two-step working scheme is proposed: (i) mass screening by ELISA for the presence of the virus; (ii) evaluation of virus infectivity within the infested seed lots. A threshold of 10 ng ml⁻¹ was determined for detection of purified ToMV by either ELISA or plant inoculation.Nicotiana tabacum cv. Xanthi NN was found to be a highly sensitive local lesion assay plant for the detection of ToMV. A positive ELISA threshold (1.3 times above the non-specific background) was set for seed samples taken from commercial seed lots by testing the same samples by both ELISA and a bioassay. http://www.phytoparasitica.org posting July 14, 2004.     

新疆番茄病毒病检测及南方番茄病毒全基因组序列测定与传播特性分析

为了解新疆番茄上病毒病的发生情况,利用一步法RT-PCR技术检测了南北疆番茄上南方番茄病毒(Southern tomato virus,STV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番茄花叶病毒(Tomato mosaic virus,ToMV)以及马铃薯Y病毒(Potato virus Y,PVY)的感染情况,并利用分段克隆的方法进行全基因组测序,通过RT-PCR方法检测健康植株与携带病毒植株杂交育种的F1代植株带毒率以分析STV的种子传播特性。结果显示,新疆番茄上CMV、STV、ToMV和PVY在北疆的检出率分别为52%、37%、27%和14%;在南疆的检出率分别为79%、60%、69%和0;且以STV、CMV及ToMV的复合侵染为主。从我国加工番茄上首次获得了长3 437 nt的STV SHZ-1核苷酸序列,序列比对分析发现其与已报道STV只有1~9个核苷酸的变异,且序列变异与地域无相关性。分析杂交F1代加工番茄植株上STV的传播特性,发现其除可由种子传播外,也可通过花粉传播。表明STV是侵染新疆番茄的主要病毒之一,且该病毒可通过种子或杂交育种途径进行传播。     

Effects of foliar sprayed calcium sources onTomato mosaic virus (ToMV) infection in tomato plants grown in greenhouses

Spray solutions containing 0.3% Ca which were prepared from four different calcium sources were foliar-sprayed on greenhouse-grown tomato plants, infected with theTomato mosaic virus Tobamovirus (ToMV) or not. ToMV-infected and uninfected control groups were sprayed with distilled water. Growth and macronutrient (N, P, K, Ca and Mg) composition of tomato plants as well as virus concentration and its relative infectivity were investigated in treated and untreated plants. The Ca sprays were applied three times: on the same day as inoculation, and 15 and 30 days after inoculation. Virus concentration in tomato plants generally decreased with foliar-sprayed Ca. Virus concentration (DAS-ELISA absorbance) was reduced by foliar-sprayed Ca, but plants remained infected. At the same time, tissue Ca concentrations increased significantly with foliar-applied Ca, with the exception of CaNO₃·4H₂O+0.05M Na-EDTA. ToMV reduced the fresh and dry weights and Ca concentrations of tomato plants, but significantly raised P concentration in the tissue. Neither virus inoculation nor foliar Ca applications affected N and Mg concentrations in tomato plants. The foliar-applied Ca from all the sources gave K concentrations similar to those of control plants. http://www.phytoparasitica.org posting Jan. 26, 2007.     

<Emphasis Type="Italic">Amaranthus leaf mottle virus</Emphasis>: 3′-end RNA sequence proves classification as distinct virus and reveals affinities within the genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Amaranthus leaf mottle virus (AmLMV) was classified as a member of the genus Potyvirus on the basis of its particle morphology, serology, and biological properties (Casetta et al., 1986). Based on these properties, an Amaranthus viridis-infecting virus isolated in Spain, causing mottle and leaf blistering as well as reduced growth has been identified as AmLMV. The 3′ terminal genomic region of this and a reference isolate from Italy has been sequenced and reveals a 95% nucleotide identity between the two isolates. The sequenced part comprises the coat protein with 281 amino acids and 315 nucleotides of the 3′ untranslated region (UTR) preceding a polyadenylated tail. Pairwise comparisons and phylogenetic analysis of the nucleotide and deduced amino acid sequences of the CP and 3′ UTR of the cloned cDNAs with those of other potyviruses shows that AmLMV is a distinct potyvirus closely related to Potato virus Y.     

<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> and <Emphasis Type="Italic">P. peruviana</Emphasis>, new natural hosts of <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>

Tomato chlorosis virus causes yellow leaf disorder epidemics in many countries worldwide. Plants of Physalis ixocarpa showing abnormal interveinal yellowing and plants of Physalis peruviana showing mild yellowing collected in the vicinity of tomato crops in Portugal were found naturally infected with ToCV. Physalis ixocarpa and P. peruviana were tested for susceptibility to ToCV by inoculation with Bemisia tabaci, Q biotype. Results confirmed that ToCV is readily transmissible to both species. The infection was expressed in P. ixocarpa by conspicuous interveinal yellow areas on leaves that developed into red or brown necrotic flecks, while P. peruviana test plants remained asymptomatic. Infected plants of both P. ixocarpa and P. peruviana served as ToCV sources for tomato infection via B. tabaci transmission. This is the first report of P. ixocarpa and P. peruviana as natural hosts of ToCV.