Complete genome sequence and construction of an infectious full-length cDNA clone of a cucumber vein yellowing virus (CVYV) isolate from Portugal.

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.

Observations on early fungal infections with relevance for replant disease in fine roots of the rose rootstock Rosa corymbifera 'Laxa'.

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.

Development of a molecular assay for the detection of Cucumber mosaic virus and the discrimination of its subgroups I and II.

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.

Complete genome sequences of two biologically distinct isolates of Asparagus virus 1.

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.

Molecular diversity of poleroviruses infecting cucurbit crops in four countries reveals the presence of members of six distinct species.

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.

Taxonomic relatedness between Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. odoriferum and Pectobacterium carotovorum subsp. brasiliense subsp. nov.

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.

Transreplication of a Tomato yellow leaf curl Thailand virus DNA-B and replication of a DNAbeta component by Tomato leaf curl Vietnam virus and Tomato yellow leaf curl Vietnam virus.

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.

Construction of an infectious full-length cDNA clone of potato virus M.

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.

Eriophyid mite transmission and host range of a Brome streak mosaic virus isolate derived from a full-length cDNA clone.

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.

Molecular characterization and detection of Vicia cryptic virus in different Vicia faba cultivars.

After extraction of double-stranded (ds) RNAs from Vicia faba, dsRNA1 and dsRNA2 of Vicia cryptic virus (VCV), a member of the genus Alphacryptovirus (family Partitiviridae), were detected in six out of seven different cultivars by agarose gel electrophoresis. In attempts to sequence the complete VCV genome, the dsRNA1 and dsRNA2 sequences from a total of five different V. faba cultivars were determined. Analysis of these sequences indicated that V. faba cultivars contain almost indistinguishable VCV sequences. The larger dsRNA1 was 2012 bp in length and contained a major open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp). The smaller dsRNA2 was 1779 bp in length and comprised a single ORF on its plus-strand encoding the coat protein (CP). The sequences of the dsRNA1 and dsRNA2 ORFs shared highest amino acid sequence identities (84 and 56%, respectively) with the corresponding gene products of the alphacryptovirus white clover cryptic virus 1 (WCCV-1). The 5'-terminal untranslated regions of dsRNA1 and dsRNA2 of VCV were highly conserved and were strikingly similar to the corresponding regions of WCCV-1. RdRp amino acid sequence alignments revealed conserved motifs, which correlate with the phylogenetic clustering of the family Partitiviridae.

Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus.

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.

The complete nucleotide sequence of a capsicum chlorosis virus isolate from Lycopersicum esculentum in Thailand.

The complete nucleotide sequence of a tospovirus isolated from Lycopersicum esculentum in Thailand was determined. The L RNA comprises of 8912 nt and codes for the RNA-dependent RNA-polymerase (RdRp) (2877 aa). Two ORFs are located on the M RNA (4823 nt) encoding the non-structural (NSm) protein (308 aa) and the viral glycoprotein precursors (Gn/Gc) (1121 aa) separated by an intergenic region of 433 nt. ORFs coding for the non-structural (NSs) and nucleocapsid (N) protein, 439 aa and 275 aa, respectively, were identified on the S RNA (3477 nt) separated by an intergenic region of 1202 nt. The N protein of the Thailand isolate was most closely related to that of capsicum chlorosis virus (CaCV), sharing an amino acid sequence identity of 92.7%. Additionally, multiple sequence analyses revealed significant similarities to tospoviruses of the species Watermelon silver mottle virus and to several putative tospovirus entries in GenBank. Based on these alignments it is proposed to refer to all these different viruses as isolates of CaCV.

Ceratothripoides claratris, a New Vector of a Capsicum chlorosis virus Isolate Infecting Tomato in Thailand.

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.

Viruses Infecting Cassava in Kenya.

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.

The complete nucleotide sequence of Plum pox virus isolates from sweet (PPV-SwC) and sour (PPV-SoC) cherry and their taxonomic relationships within the species.

Plum pox virus (PPV) sweet (SwC) and sour (SoC) cherry isolates were the first PPV isolates to be recovered from natural infection in sweet and sour cherry plants, respectively. Their complete nucleotide sequences have been determined finding a deduced genome organisation typical for PPV species. Both genomes are 9795 nucleotides long, excluding the 3' terminal poly(A) tail, and contain an open reading frame of 9432 nt, encoding a polyprotein of 3143 amino acids. The nucleotide and predicted amino acid sequences of PPV-SwC and SoC have been pairwise compared with available sequences of different PPV strains. Although a very high similarity exists between the whole genomes and polyproteins of the two cherry isolates, high levels of divergence have been calculated with sequences of PPV-M, D and EA isolates. In particular, the most considerable divergence has been found in part of 5' non coding region, in regions encoding P1, P3 + 6K1, 6K2 and NIa-VPg proteins as well as in the N-terminal domain of the coat protein. Phylogenetic analysis have been undertaken in order to establish the taxonomic localisation of SwC and SoC isolates within PPV species, showing that they are always clustered together and separated from the rest of PPV strains, being clearly the most distant.

Multiplex RT-PCR-ELISA compared with bioassay for the detection of four apple viruses.

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.

The complete sequence of the genome of Cocksfoot streak virus (CSV), a grass infecting Potyvirus.

The complete nucleotide sequence of Cocksfoot streak virus (CSV) has been determined. The viral genome comprises 9663 nucleotides, excluding a 3'-terminal poly(A) sequence. The genome of CSV has a 133 nt 5'-non coding and a 260 nt 3'-non coding region. The RNA of CSV encodes a single polyprotein of 3089 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae. CSV is transmissible by aphids and has a narrow host range in the Gramineae. It was compared to two potyviruses having monocotyledonous hosts (monocot potyviruses) and several potyviruses infecting dicotyledonous plants (dicot potyviruses). CSV is most closely related to other monocot potyviruses like Maize dwarf mosaic virus (MDMV) and Johnson grass mosaic virus (JGMV), but also closely related to the dicot potyviruses. On the other hand, CSV is less related to monocot viruses from the other genera (Rymovirus, Tritimovirus) within the Potyviridae. Specific motifs, described for potyviral polyproteins, are also present in the polyprotein of CSV. Only two motifs in the HC-Pro--motif involved in long distance movement and motif for HC-Pro self-interaction--were different in comparison to most of the dicot potyviruses.

Molecular and serological relationships of Spartina mottle virus (SpMV) strains from Spartina spec. and from Cynodon dactylon to other members of the Potyviridae.

Spartina mottle virus (SpMV) was first reported 1980 and classified by physical and biological properties as a tentative member of the genus Rymovirus in the Potyviridae. This genus was recently separated into two genera: Rymovirus and Tritimovirus. Now the sequence of the 3'-terminal part of the genome of SpMV was determined. Additionally a virus isolate originating from Cynodon dactylon in Italy was cloned and sequenced. This Assisi-isolate shared 87.5% amino acid sequence identity with SpMV. The high degree of identity and their close serological relationship indicate that SpMV and Assisi-isolate have to be regarded as different strains of one virus. The Assisi-isolate should be designated as SpMV-AV. Comparing the C-terminal part of the ORF of several Potyviridae the sequences of SpMV strains were more similar to those of the genera Rymovirus and Potyvirus than to the genera Tritimovirus, Macluravirus, Bymovirus and Ipomovirus. The comparisons revealed identities of less than 32% for the CP and 37% for the 3'-NIb/CP region, indicating that SpMV can not be classified to any of the established genera. The results of serological tests support a separate position of SpMV in the Potyviridae. We propose to introduce the name Sparmovirus for the new genus.