RESUMEN
Cucumber vein yellowing virus (CVYV) is a member of the genus Ipomovirus in the family Potyviridae. In the National Center for Biotechnology Information (NCBI) database, three complete genome sequences of CVYV isolates from Spain (NC_006941), Israel (KT276369), and Jordan (JF460793) are available. In this study, we report the complete sequence of an isolate of CVYV from Portugal (DSMZ PV-0776) along with the construction of an infectious full-length cDNA clone via Gibson assembly. The sequence of CVYV Portugal shows the closest relationship to a CVYV isolate from Spain (genome, 99.7% identity; polyprotein, 99.7% identity). The CVYV full-length cDNA clone was introduced by electroporation into Rhizobium radiobacter and infiltrated into the cotyledons of Cucumis sativus plantlets, resulting in symptoms resembling those of the wild-type virus. Transmission of the infectious CVYV full-length clone by the whitefly Bemisia tabaci was confirmed. This first report confirming the infectivity of a CVYV cDNA clone provides the opportunity to study gene functions in a consistent genomic background.
Asunto(s)
Cucumis sativus , Células Clonales , ADN Complementario/genética , Enfermedades de las Plantas , Portugal , PotyviridaeRESUMEN
The complete genome sequences of two asparagus virus 1 (AV-1) isolates differing in their ability to cause systemic infection in Nicotiana benthamiana were determined. Their genomes had 9,741 nucleotides excluding the 3'-terminal poly(A) tail, encoded a polyprotein of 3,112 amino acids, and shared 99.6 % nucleotide sequence identity. They differed at 37 nucleotide and 15 amino acid sequence positions (99.5 % identity) scattered over the polyprotein. The closest relatives of AV-1 in amino acid sequence identity were plum pox virus (54 %) and turnip mosaic virus (53 %), corroborating the classification of AV-1 as a member of a distinct species in the genus Potyvirus.
Asunto(s)
Asparagus/virología , Chenopodium quinoa/virología , Genoma Viral/genética , Nicotiana/virología , Enfermedades de las Plantas/virología , Potyvirus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/virología , Virus Eruptivo de la Ciruela/genética , Potyvirus/aislamiento & purificación , ARN Viral/genética , Análisis de Secuencia de ARN , Homología de Secuencia de Ácido Nucleico , Tymovirus/genética , Proteínas Virales/genéticaRESUMEN
When 66 cucurbit samples with yellowing symptoms from fields in Mali, the Philippines, Thailand and Uzbekistan were screened by RT-PCR using universal polerovirus primers, 21 were identified as harboring polerovirus RNA. When these 21 samples were screened with specific primers for the known cucurbit-infecting poleroviruses, suakwa aphid-borne yellows virus and a recombinant strain of cucurbit aphid-borne yellows virus were detected for the first time in the Philippines and Thailand. However, seven polerovirus-positive samples did not react with any of the known species-specific primers. Sequencing of 1.4-kb universal polerovirus RT-PCR products revealed the presence of two poleroviruses that had not been described previously. These viruses, from Mali and Thailand, were provisionally named pepo aphid-borne yellows virus and luffa aphid-borne yellows virus, respectively.
Asunto(s)
Cucurbita/virología , Variación Genética , Luteoviridae/clasificación , Luteoviridae/aislamiento & purificación , ARN Viral/genética , Asia , Análisis por Conglomerados , Genotipo , Luteoviridae/genética , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
AIMS: Pectobacterium carotovorum is a heterogeneous species consisting of two named subspecies, P. carotovorum subsp. carotovorum and P. carotovorum subsp. odoriferum. A third subspecies, P. carotovorum subsp. brasiliense, was previously proposed. The study aimed to confirm the subspecies status and validate the proposed name of P. carotovorum subsp. brasiliense using a novel and standard microbial taxonomy. METHODS AND RESULTS: DNA-DNA hybridization confirmed that P. carotovorum subsp. brasiliense is a different species from P. wasabiae, P. betavasculorum and P. atrosepticum, with 28, 35 and 55% similarity values, respectively, but is a member of the P. carotovorum species with 73-77% similarity values. Sequencing the entire 16S rRNA gene of two polymorphic copies from strains of each of the P. carotovorum subspecies demonstrated that the average 16S rRNA gene sequence diversity between P. carotovorum subsp. brasiliense and P. carotovorum subsp. carotovorum was lower than the maximum genetic distances between two sequence types obtained from the same strain. Multilocus sequence analysis based on eight housekeeping genes (mtlD, acnA, icdA, mdh, pgi, gabA, proA and rpoS) differentiated the subspecies and delineated two P. carotovorum subsp. brasiliense clades. CONCLUSION: Pectobacterium carotovorum subsp. brasiliense clade I was comprised of strains isolated from Brazil and Peru, while clade II included strains from Asia, North America and Europe. Strains in clade I but not clade II were phenotypically consistent with the original description of P. carotovorum subsp. brasiliense in that they produced reducing substances from sucrose and acid from α-methyl glucoside. The type strain for P. carotovorum subsp. brasiliense 212(T) (= LMG2137(T) = IBSBF1692(T) = CFBP6617(T) ) was previously designated. The GC mol content of the type strain is 51·7%. SIGNIFICANT AND IMPACT OF THE STUDY: the study introduces a full description for the strains belonging to the two different clades assigned to P. carotovorum subsp. brasiliense.
Asunto(s)
Pectobacterium carotovorum/clasificación , Filogenia , Asia , Técnicas de Tipificación Bacteriana , Composición de Base , Brasil , ADN Bacteriano/genética , Europa (Continente) , Ácidos Grasos/análisis , Genes Bacterianos , Tipificación de Secuencias Multilocus , América del Norte , Hibridación de Ácido Nucleico , Pectobacterium carotovorum/genética , Perú , Quinonas/análisis , ARN Ribosómico 16S/genéticaRESUMEN
Replant disease is a worldwide phenomenon affecting various woody plant genera and species, especially within the Rosaceae. Compared to decades of intensive studies regarding replant disease of apple (ARD), the replant disease of roses (RRD) has hardly been investigated. The etiology of RRD is also still unclear and a remedy desperately needed. In greenhouse pot trials with seedlings of the RRD-sensitive rootstock Rosa corymbifera 'Laxa' cultured in replant disease affected soils from two different locations, early RRD symptom development was studied in fine roots. In microscopic analyses we found similarities to ARD symptoms with regards to structural damages, impairment in the root hair status, and necroses and blackening in the cortex tissue. Examinations of both whole mounts and thin sections of fine root segments revealed frequent conspicuous fungal infections in association with the cellular disorders. Particularly striking were fungal intracellular structures with pathogenic characteristics that are described for the first time. Isolated fungi from these tissue areas were identified by means of ITS primers, and many of them were members of the Nectriaceae. In a next step, 35 of these isolates were subjected to a multi-locus sequence analysis and the results revealed that several genera and species were involved in the development of RRD within a single rose plant. Inoculations with selected single isolates (Rugonectria rugulosa and Ilyonectria robusta) in a Perlite assay confirmed their pathogenic relationship to early necrotic host plant reactions, and symptoms were similar to those exhibited in ARD.
Asunto(s)
Hypocreales/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Raíces de Plantas , Rosa , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Rosa/metabolismo , Rosa/microbiologíaRESUMEN
The genomes of two tomato-infecting begomoviruses from Vietnam were cloned and sequenced. A new variant of Tomato leaf curl Vietnam virus (ToLCVV) consisting of a DNA-A component and associated with a DNAbeta molecule as well as an additional begomovirus tentatively named Tomato yellow leaf curl Vietnam virus (TYLCVV) consisting also of a DNA-A component were identified. To verify if monopartite viruses occurring in Vietnam and Thailand are able to transreplicate the DNA-B component of Tomato yellow leaf curl Thailand virus-[Asian Institute of Technology] (TYLCTHV-[AIT]) infectivity assays were performed via agroinoculation and mechanically. As result, the DNA-B component of TYLCTHV-[AIT] was transreplicated by different DNA-A components of viruses from Vietnam and Thailand in Nicotiana benthamiana and Solanum lycopersicum. Moreover, the TYLCTHV-[AIT] DNA-B component facilitated the mechanical transmission of monopartite viruses by rub-inoculation as well as by particle bombardment in N. benthamiana and tomato plants. Finally, defective DNAs ranging from 735 to 1457 nucleotides were generated in N. benthamiana from those combinations containing TYLCTHV-[AIT] DNA-B component.
Asunto(s)
Begomovirus/fisiología , Replicación del ADN , ADN Viral/biosíntesis , Secuencia de Bases , Begomovirus/aislamiento & purificación , Clonación Molecular , ADN Viral/química , ADN Viral/genética , Prueba de Complementación Genética , Genoma Viral , Solanum lycopersicum/virología , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia , Nicotiana/virología , VietnamRESUMEN
A nucleic acid based test for the detection of the economically important plant virus Cucumber mosaic virus (CMV) based on the Luminex xTAG technology was developed. This technology has the advantage of allowing the simultaneous detection of various targets. Applying this method, we prove the presence of CMV in general and differentiate between its two subgroups I and II for which significant differences concerning severity of symptoms and virulence have been reported. For the development of the test procedure the coat protein gene sequences of 29 CMV isolates were cloned, sequenced and classified into subgroups. Sequences from GenBank were used to design primers. Additionally, a subgroup specific ELISA was conducted for comparison. This work is part of a project which aims to develop a test for the simultaneous detection of various plant pathogens (viral, bacterial and fungal) in plant material.
Asunto(s)
Cucumovirus/clasificación , Cucumovirus/aislamiento & purificación , Técnicas de Genotipaje/métodos , Técnicas de Diagnóstico Molecular/métodos , Enfermedades de las Plantas/virología , Proteínas de la Cápside/genética , Cucumovirus/genética , Cartilla de ADN/genética , Análisis de Secuencia de ADNRESUMEN
ABSTRACT Ceratothripoides claratris, the predominant thrips species on tomato in Thailand, was tested for vector competence and efficiency to transmit Capsicum chlorosis virus (CaCV) (isolate AIT) to tomato. The efficiency of adult-stage transmission was influenced by the larval stage at which virus was acquired. Adult C. claratris showed 69% transmission efficiency after acquiring the virus as freshly emerged (<1 h) first-instar larvae. However, when just molted (<1 h) second-instar larvae acquired the virus, the percentage of adult transmitters significantly decreased (48%). Transmission efficiency of up to 47% was detected with second-instar larvae of C. claratris which had acquired the virus as freshly emerged first-instar larvae. Transmission efficiency did not significantly differ between adult males and females, irrespective of the larval stage at which the virus was acquired. Highest transmission efficiency for CaCV was recorded in adult C. claratris derived from second-instar larvae collected from infected tomato plants in a greenhouse. Lowest transmission efficiency was observed in adults directly collected from infected tomato plants in the greenhouse. The spread of CaCV on tomato plants in greenhouses showed a close association with thrips infestations.
RESUMEN
The 25K movement protein (MP) of potato virus X (PVX) is encoded by the 5'-proximal gene of three overlapping MP genes forming a 'triple gene block'. The PVX 25K MP (putative NTPase-helicase) has been synthesized in Escherichia coli as a recombinant containing a six-histidine tag at the amino terminus. The His-tagged 25K protein was purified in a one-column Ni-chelate affinity chromatography procedure. In the absence of any other viral factors, this protein had obvious Mg2+-dependent ATPase activity, which was stimulated slightly (1.7-1.9-fold) by various polynucleotides. Like other viral proteins possessing ATPase-helicase motifs and many plant viral movement proteins, the PVX 25K MP was able to bind nucleic acids in vitro. The RNA binding activity of the 25K MP was pronounced only at very low salt concentrations and was independent of its ATPase activity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Potexvirus , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Proteínas Virales/metabolismo , Ácido Anhídrido Hidrolasas/metabolismo , Secuencia de Aminoácidos , Escherichia coli/genética , Datos de Secuencia Molecular , Peso Molecular , Nucleósido-Trifosfatasa , Proteínas de Movimiento Viral en Plantas , ARN Helicasas , ARN Nucleotidiltransferasas/metabolismo , ARN Viral/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
From sequence comparisons between the tobramovirus genomes an open reading frame (ORF-X) potentially encoding a small, positively charged protein (33- to 45-amino-acids long) was found to overlap the immediate 3' and 5' sides of the transport protein gene and coat protein gene, respectively. In vitro translation of the monocistronic artificial transcripts generated with T7 RNA polymerase yielded a protein of M(r) 4000 (p4) and an unexpected trypsin-sensitive complex of M(r) 54,000 that was resistant to reduction with 2-mercaptoethanol but could be dissociated by 8 M urea. Assembly of this complex was inhibited completely by site-directed mutagenesis within a conserved, positively charged 5-amino-acid long segment of the ORF-X protein. After centrifugation in low salt buffer the 54-kDa complex remained mostly associated with ribosomes. Apparently this complex represents a specific aggregate of the p4 product of ORF-X with a protein of approximate M(r) 50,000 that is a component of the translation apparatus.
Asunto(s)
Virus del Mosaico del Tabaco/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sistema Libre de Células , ADN Viral , ARN Polimerasas Dirigidas por ADN , Electroquímica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
The serological relationships among strains of bean common mosaic virus (BCMV) (genus Potyvirus, family Potyviridae) were investigated by testing 13 isolates of the 10 known BCMV pathotypes with two monoclonal antibodies and six antisera to BCMV strains. In addition, other properties of serologically distinct BCMV strains were compared. Two groups of BCMV strains were obtained by ELISA and Western blot serology: serotype A contained the BCMV strains NL3, NL5, and NL8 and serotype B contained the BCMV strains NL1, NL2, NL4, NL6, US4, NL7, NY15, and Fla. SDS polyacrylamide gel electrophoresis and Western blotting of freshly purified preparations, and of extracts from leaves infected with eleven BCMV strains showed that the apparent molecular mass of the capsid protein of the serotype A isolates NL3, NL5, and NL8 are lower (about M(r) 33,000) than those of the serotype B isolates (M(r) 34,500 to 35,000). The normal lengths of the particles of the serotype A isolates were shorter (810-818 nm) than those of most isolates (except NL6 and NY15) of serotype B (847-886 nm). All isolates studied induced cytoplasmic pinwheel and scroll inclusions. Cells infected with serotype A isolates contained a specific type of proliferated endoplasmic reticulum which was never found in cells infected with serotype B isolates. The capsid protein gene of a representative member of each serotype was cloned and sequenced. Molecular mass calculations based upon nucleotide sequence-derived amino acid sequences yielded M(r) of 29,662 and 32,489 for the capsid proteins of the serotype A isolate NL8 and the serotype B isolate NL4, respectively. Comparison of the coat-protein sequences showed considerable differences at the N-termini whereas the core regions and the C-termini appeared to be highly conserved. Marked differences were also observed within the 3' non-coding regions of cloned cDNAs of NL 4 and NL 8. The striking differences between the two serotypes of BCMV strongly suggest that they be classified as two distinct potyviruses which naturally infect Phaseolus beans.
Asunto(s)
Virus del Mosaico/clasificación , Virus ARN/clasificación , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Cápside/química , Cápside/genética , Efecto Citopatogénico Viral , ADN Viral , Epítopos/inmunología , Fabaceae/microbiología , Intrones , Datos de Secuencia Molecular , Peso Molecular , Virus del Mosaico/ultraestructura , Plantas Medicinales , Virus ARN/ultraestructura , Conejos , Homología de Secuencia de Aminoácido , Serotipificación , Especificidad de la EspecieRESUMEN
Two multiplex RT-PCR assays with specific coamplification of plant mRNA as an internal control from total nucleic acids are described for the parallel detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus and Apple stem grooving virus. All are important economically and common pathogens in commercial apple and pear cultivars, except for Apple mosaic virus. Four virus specific primer pairs and one primer pair which allows the specific amplification of mRNA of the mitochondrial nad5 gene are described. Specificity of all primer pairs was confirmed by sequencing the RT-PCR products. A range of different virus isolates from various geographic origins could be detected by these multiplex RT-PCR assays all year round. Viruses were detected reliably in composite extracts at a ratio of one part total nucleic acid extract from an infected sample mixed with 39 parts of extract from healthy samples. The use of the internal control minimizes the risk of obtaining false negative RT-PCR results, which is desirable for routine testing, and avoids the need to eliminate contaminating DNA in extracts. To our knowledge, this is the first report on the use of a specific internal RNA control from total nucleic acids. The multiplex RT-PCR assays described are reliable, rapid and sensitive methods for the detection of these viruses, and may replace techniques need commonly like indexing by woody indicators or ELISA.
Asunto(s)
Malus/virología , Enfermedades de las Plantas/virología , Virus de Plantas/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Malus/genética , Virus de Plantas/genética , ARN Mensajero/genética , ARN de Planta/genética , ARN Viral/análisis , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
A sensitive and reliable multiplex RT-PCR-ELISA technique for the detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus and Apple stem grooving virus was developed. This technique is compared with the method used commonly for indexing by woody indicators, which is time consuming and expensive. For the RT-PCR-ELISA technique, the amplified products were labeled with digoxigenin during the RT-PCR by incorporation of a digoxigenin labeled primer. After hybridization of the PCR products to specific capture oligonucleotides, which were bound covalently to the surface of NucleoLink strips, anti-digoxigenin antibodies were used for detection. More than 100 samples were tested in parallel by indexing and multiplex-RT-PCR-ELISA. All infections detected by woody indicators were also detected by multiplex RT-PCR-ELISA. Furthermore, additional infections were only found by multiplex RT-PCR-ELISA. The colourimetric detection of multiplex-RT-PCR products was at least as sensitive and sometimes slightly more sensitive than detection by gel electrophoresis. The results show that this molecular technique is more reliable for the detection of the above mentioned apple viruses than indexing by woody indicators, thereby helping to reduce cost and time during the certification of plant material.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Malus/virología , Virus ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Bioensayo , Bromoviridae/clasificación , Bromoviridae/aislamiento & purificación , Enfermedades de las Plantas/virología , Virus de Plantas/clasificación , Virus de Plantas/aislamiento & purificación , Virus ARN/clasificación , Sensibilidad y EspecificidadRESUMEN
A survey of cassava viruses was conducted in major cassava-growing regions of Kenya. A total of 185 leaf samples and 62 stem cuttings from plants with viral disease symptoms were collected and analyzed by biological, electron microscopy, enzyme-linked immunosorbent assay, and polymerase chain reaction. All samples from western Kenya had cassava begomoviruses (African cassava mosaic virus [ACMV], East African cassava mosaic virus [EACMV], and Uganda variant [EACMV-UG]) in either single or in mixed infection. However, all samples from the Coast region were infected with only EACMV, a begomovirus. In addition, 15 samples had mixed infections of EACMV and three other hitherto unidentified filamentous viruses. The viruses observed were 200, 500, 650, and 750 nm long, respectively. In addition to rod-shaped and some flexuous viruses, as seen in a crude sap preparation, pinwheels also were observed, indicating a possible association of some of the viruses with the Potyviridae family. The symptoms induced by these viruses in Nicotiana benthamiana were very severe and often caused about 50% death of the test plants. Back inoculation onto cassava resulted in 100% infections. This finding provides evidence that, other than begomoviruses that cause serious diseases of cassava in Africa, filamentous viruses also are present and, despite their limited distribution, they could reach local significance and, most probably, be as serious as begomoviruses. The implications of these findings are discussed and recommendations for future work suggested.
RESUMEN
An infectious full-length cDNA clone of potato virus M (PVM) was produced. Total RNA was extracted from PVM-infected Nicotiana hesperis plants and used for cDNA synthesis. Subsequent RT-PCR produced two DNA fragments of about 5.5 and 3.2 kbp, which were ligated downstream of an enhanced 35S cauliflower mosaic virus promoter. After cloning of the enhanced 35S promoter with the PVM sequence into a modified pBIN19 plasmid and electroporation of Agrobacterium tumefaciens, the agroinoculated PVM full-length clone (pPVM-flc) led to systemic PVM infections in different host plants, causing symptoms indistinguishable from those caused by wild-type PVM.
Asunto(s)
Carlavirus/genética , Clonación Molecular , ADN Complementario/genética , ARN Viral/genética , Agrobacterium tumefaciens/genética , Electroporación , Orden Génico , Enfermedades de las Plantas/virología , Plantas/virologíaRESUMEN
Biolistic inoculation of Hordeum vulgare and Phalaris paradoxa with a brome streak mosaic virus (BrSMV) full-length cDNA clone (pBrSMV(fl)) led to typical leaf streak symptoms in both plant species. Infected H. vulgare plants showed a more stunted growth 8 weeks after symptom appearance compared to BrSMV wild type (BrSMV(wt))-infected plants. Moreover, a slightly higher virus titer was observed in BrSMV(fl)-inoculated H. vulgare, A. sativa and P. paradoxa plants. The biological activity of BrSMV(fl) and BrSMV(wt) was verified in vector transmission assays, providing the first experimental evidence that Aceria tosichella can act as a natural vector of BrSMV.
Asunto(s)
Vectores Arácnidos/virología , Bromovirus/fisiología , ADN Complementario/análisis , Hordeum/virología , Ácaros/virología , Animales , Biolística/métodos , Bromovirus/genética , Phalaris/virología , Enfermedades de las Plantas/virologíaRESUMEN
Tomato plants grown in greenhouses in Thailand developed typical symptoms of a tomato yellow leaf curl Thailand virus (TYLCTHV) infection. After confirmation by ELISA, a Phi29 DNA polymerase approach was chosen for further molecular analysis of TYLCTHV. Total DNA purified from infected tomato leaves was subjected to rolling-circle amplification (RCA) of DNA-A and DNA-B of TYLCVTHV. In addition, a new monopartite geminivirus with a putative recombinant background was identified by RCA and tentatively named tobacco leaf curl Thailand virus (TbLCTHV). To confirm the composition of both geminiviruses, full-length clones were established and used for inoculation of Nicotiana benthamiana by particle bombardment or agroinfection. When TYLCTHV DNA-A and DNA-B were applied together by particle bombardment or agroinfection, severe stunting, yellowing, and leaf curling were observed. Whereas TYLCTHV DNA-A and TbLCTHV revealed no infection after'particle bombardment, similar symptoms in N. benthamiana, like leaf upward curling and yellowing were observed following agroinfection.DNA components of TYLCTHV DNA-A and DNA-B were excised from their respective plasmids, ligated, and amplified by Phi29 DNA polymerase. The ability of viral concatamere inoculation was evaluated in particle co-bombardment experiments on N. benthamiana. Thus, particle bombardment of RCA-derived multimeric products proved to be at least as effective as inoculation with a partial repeat construct and tenfold as effective as inoculation with excised unit-lengths of DNA-A and DNA-B of TYLCVTHV when using each DNA component in an amount of 5 ng.