In situ hybridization and promoter analysis reveal that cotton leaf curl Multan betasatellite localizes in the phloem.

Begomoviruses (the family Geminiviridae) have either a monopartite or a bipartite (DNA A and DNA B) single-stranded DNA genome. DNA B contains the open reading frame for movement protein and enables some bipartite begomoviruses to invade non-phloem tissues, whereas monopartite begomoviruses are limited to the phloem. Betasatellite DNA replicates in association with some monopartite begomoviruses to produce severe symptoms and enhance replication of helper virus in some hosts. The cotton leaf curl Multan betasatellite (CLCuMuB) has been shown to substitute for the DNA B of a bipartite begomovirus by permitting systemic infection, but the tissue tropism and the role of betasatellites in releasing monopartite begomoviruses from the phloem to adjacent tissue has not been described. In this study, the tissue tropism of CLCuMuB and a monopartite helper virus, tomato leaf curl virus (ToLCV), has been investigated using in situ hybridization, promoter localization and analysis of transgenic 2ß plants, which contain a dimer of the CLCuMuB genome. In situ hybridization showed that CLCuMuB and ToLCV were localized into the vascular tissue of infected plants. In addition, CLCuMuB was detected only in the vascular tissue of 2ß transgenic plants after infection with ToLCV. ß-glucuronidase (GUS) expression under the ßC1 promoter was also limited to the phloem tissues. In conclusion, we showed for the first time that CLCuMuB localizes only in the phloem tissues and unlike the DNA B component, CLCuMuB is unable to release the monopartite helper virus out of phloem tissues.

Current status of viroid taxonomy.

Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.

Nucleotide sequence diversity in Velvet tobacco mottle virus: a virus with a unique Australian pathosystem.

Velvet tobacco mottle virus (VTMoV) is a naturally occurring mirid-transmitted sobemovirus of native velvet tobacco (Nicotiana velutina) plants in the Australian arid zone. We have sequenced the coding region of a typical field isolate of VTMoV (isolate I-17-04, satellite-plus) and show that it differed by nine polymorphisms from the previously sequenced atypical 'satellite-minus' variant VTMoV-K1 (represented here as L-K1-04), while retaining the same genomic and amino acid sequence motifs. We also report that although L-K1-04 was confirmed to be free of detectable satellite RNA by gel electrophoretic assay, the satellite sequence was detected in it by RT-PCR assay. Nucleotide sequence variation among the RNA-dependent RNA polymerase open reading frames of 15 field and laboratory isolates identified four phylogenetic groups, but these did not show a pattern related to site or time of sampling. This result would be consistent with nucleotide sequence variants of VTMoV being dispersed widely by migrating adult mirid vectors.

Mutation rate in Velvet tobacco mottle virus varies between genomic region and virus variant but is not influenced by obligatory mirid transmission.

Genomic mutation in plant viruses of cultivated plants is known to be influenced by virus, host and vector, but the factors influencing mutation in viruses of native plants in natural ecosystems are rarely studied. We have tested the effect of mode of transmission on mutation in Velvet tobacco mottle virus (VTMoV), a mirid-vectored sobemovirus associated with Nicotiana velutina, an Australian native xerophyte growing in a region isolated from anthropogenic influences. Two variants of VTMoV (K1 and R17) were passaged monthly in the alternative experimental plant host, N. clevelandii, for 2 years, either by mechanical inoculation or by transmission with the mirid Cyrtopeltis nicotianae. Sequence variations were scored after 24 passages in regions of the genome containing the open reading frames (ORFs) for the P1 and coat protein (CP). The mean mutation rate was 6.83 × 10(-4) nt/site year, but a higher overall rate was observed for the K1 (satellite -) than the R17 (satellite +) variant. The P1 ORF showed a higher frequency of non-synonymous mutations than the CP. No clear association was found between either mutation site or mutation rate and the mode of transmission, indicating that obligatory mirid transmission had not exerted a specific bottle-neck effect on sequence variation during the experimental time frame. Failure to detect any sequence motifs linked to vector transmission suggests that a specific capsid-stylet interaction is not required for transmission by mirids.

Complete nucleotide sequence of Velvet tobacco mottle virus isolate K1.

Velvet tobacco mottle virus (VTMoV) infects the native Australian plant Nicotiana velutina, which is endemic to central Australia. This virus is included in the genus Sobemovirus based on virion morphology and serological relationships. We report here the full genome sequence of VTMoV, attained using a genome-walking strategy with both degenerate and specific primers. This sequence confirms that VTMoV is a sobemovirus, with the same open reading frame (ORF) organisation as other described sobemoviruses. The VTMoV sequence is closest to those sobemoviruses isolated from monocotyledonous plants, although the narrow host range of VTMoV is limited to dicotyledonous plants.

Discriminating Between Isolates of PSbMV Using Nucleotide Sequence Polymorphisms in the HC-Pro Coding Region.

The molecular diversity of 14 isolates of Pea seed-borne mosaic virus (PSbMV) from southern Australia, 13 previously described isolates from Pakistan, and a reference isolate from the United States have been studied to determine whether a relatively simple molecular diagnostic assay and classification scheme could be developed for this virus. The Australian isolates were placed into either pathotype P1 or pathotype P4 by bioassay on differential genotypes of Pisum sativum. The Pakistani isolates represented pathotypes P1, P4, U1, and U2, and an undetermined pathotype. The reference US isolate was pathotype P1. A reverse transcription-polymerase chain reaction (RT-PCR) assay based on an amplicon from the variable HC-Pro coding region of potyviruses was shown to distinguish PSbMV from seven other legume infecting potyviruses. Restriction fragment length polymorphisms (RFLPs) generated from the HC-Pro RT-PCR products of all 28 isolates using seven restriction endonucleases placed them into eight groups. A phylogenetic tree based on a Bray-Curtis similarity comparison placed the groups into three clusters. The groups and clusters had no clear association with either pathotype or geographic source. It is concluded that within the range of viruses and isolates tested, the RT-PCR-RFLP method will both specifically identify PSbMV and provide a simple, qualitative, and rapid means for placing PSbMV isolates into groups. Applications could include mapping and tracking isolates in space and time.

Single antibody dot immunoassay--a simple technique for rapid detection of a plant virus.

A single antibody dot immunoassay (SADI) was developed for use as a rapid, simple and sensitive technique for the direct assay of virus bound to nitrocellulose membranes. SADI has been tested for the detection of subterranean clover mottle virus (SCMoV) in small amounts of infected tissue and purified virus preparations, and the technique was found to be twelve times more sensitive than ELISA in terms of total antigen detected. DIBA, an indirect dot immunoassay, was about twice as sensitive as SADI, but the latter was more specific for the detection of SCMoV and is a simpler, more rapid assay, requiring less than three h to complete.

Detection of an unidentified potyvirus from Roystonea regia palm using the polymerase chain reaction and degenerate, potyvirus specific, primers and potential problems arising from the amplification of host plant DNA sequences.

Degenerate potyvirus-specific primers were used in the PCR to amplify cDNA representing a 335 nucleotide region of the coat protein gene in RNA purified from an uncharacterised potyvirus isolated from Roystonea regia palms in Queensland, Australia. The RNA was also detected by PCR in total nucleic acid extracts from infected Nicotiana benthamiana, N. clevelandii and Vicia faba. The method also successfully detected pea seed-borne mosaic virus in Pisum sativum. In addition the procedure amplified DNA's of approximately 200 bp and 420 bp, of non-viral origin, from total nucleic acid extracts of healthy Nicotiana spp, indicating that size of the PCR products needs to be included as a criterion for identifying virus specific products when this method is used.

Conformational transitions in viroids and virusoids: comparison of results from energy minimization algorithm and from experimental data.

Viroids are single-stranded circular RNA molecules of 240 to 400 nucleotides which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but replicate only as genomic part of a plant virus. Their structure and structural transitions have been investigated by thermo-dynamic, kinetic and hydrodynamic methods. The special features of the sequences of these RNAs, which are the basis for their secondary structures and structural flexibility, are investigated with theoretical methods. A set of thermodynamic parameters for helix growth and loop formation is selected from the literature to calculate secondary structures and structural transitions of single-stranded RNAs. Appropriate modifications of the chosen parameter set are discussed. For calculations we used either Tinoco-plots and the model of "cooperative helices" or the Zuker-program based on the exact algorithm of Nussinov et al, or both. Calculations were done for viroids and virusoids. As both are single-stranded, circular RNAs we had to modify the Zuker-program as described in the appendix. Calculations are done for different viroids, i.e. potato spindle tuber, citrus exocortis, chrysanthemum stunt, coconut cadang-cadang, and avocado sunblotch, and for two virusoids, i.e. the circular RNAs of Solanum nodiflorum mottle virus, and velvet tobacco mottle virus. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but statistical sequences. The comparison of viroids, virusoids, and circular RNA or random sequences confirms the uniqueness of viroid structure.

Specific identification of coconut tinangaja viroid for differential field diagnosis of viroids in coconut palm.

ABSTRACT Tinangaja is a widespread lethal disease of putative viroid etiology affecting coconut palm on the island of Guam. Determination of its distribution and mode of spread requires a simple and reliable diagnostic procedure that is specific for the associated coconut tinangaja viroid (CTiVd). A method of extracting tissue followed by analytical agarose gel electrophoresis for CTiVd detection has been developed and used to identify the viroid in leaf samples of suspect symptomatic palms growing in the field. Two-dimensional polyacrylamide gel electrophoresis showed that the viroid band contained circular molecules that are typical for viroids. Confirmation of the identity of CTiVd and detection of low levels of viroid below the threshold of detection by agarose gel electrophoresis was achieved either by diagnostic oligonucleotide-probe (DOP) hybridization assay or by reverse-transcription polymerase chain reaction (RT-PCR) with the oligonucleotide probe as one of the two PCR primers. RT-PCR was not substantially more sensitive than DOP-hybridization assay. This procedure also was applicable to coconut cadang-cadang viroid (CCCVd), and oligonucleotide probes designed to be specific for either CTiVd or CCCVd distinguished between these two viroids in coconut leaf extracts. This strategy provides a rapid and specific indexing procedure for the two characterized viroids of coconut palm and will be applicable to further studies on the viroid-like sequences previously reported in tropical monocotyledons.

Identification of sugarcane striate mosaic-associated virus by partial characterization of its double-stranded RNA.

ABSTRACT Sugarcane striate mosaic (ScSM)-affected sugarcane leaves contain a disease-associated 9-kilobase (kb) double-stranded RNA (dsRNA), usually together with 6- and 2.6-kb dsRNAs. The purified 9-kb dsRNA was amplified by the randomly primed polymerase chain reaction (PCR) and cloned. The nucleotide sequences of three separate regions, representing about 2.55 kb (28%) of the dsRNA sequence, were found to have significant similarities to viruses in the genera Capillo-, Carla-, Fovea-, Potex-, Poty-, Tricho-, and Tymovirus. Greatest overall similarity was found to apple stem pitting virus, with less similarity to blueberry scorch virus and potato virus M. A standard virus purification procedure was used to identify slightly flexuous filamentous particles that copurified with the disease-associated RNA. Particle modal lengths were approximately 950 and 1,900 nm with a diameter of 15 nm. Preparations contained a 51-kDa putative capsid protein and a 9-kb single-stranded RNA with a probable 3' polyadenylate tract. These ScSM-associated virus particles differ physically from viruses in existing genera because of their relative rigidity, length, and putative coat protein size. Reverse-transcription PCR with a primer pair designed from the sequenced segments amplified a 820-base pair fragment from ScSM-affected but not healthy sugarcane plants.

Early Season Survey of Pea Viruses in Pakistan and the Detection of Two New Pathotypes of Pea Seedborne Mosaic Potyvirus.

Sixty-two commercial pea fields or experimental plots located in eight districts of the major pea-growing areas of North West Frontier Province (NWFP) of Pakistan were surveyed for pea viruses in the early winter growing season of 1995. Samples were analyzed by dot-immunobinding assay (DIBA) using antisera to 14 different viruses. Of the 713 plants sampled, 82 were positive for either bean yellow mosaic potyvirus, cucumber mosaic cucumovirus, or pea seedborne mosaic potyvirus (PSbMV), with an average incidence of 9.4, 0.57, and 1.5%, respectively. PSbMV was also detected in 1 to 5% of dry seed from five of the 12 pea varieties tested and in 8 to 20% of seedlings raised from seed of three of these varieties. The infectivities of 12 PSbMV isolates found in the survey of pea varieties from Pakistan were compared using a standard range of pea differential genotypes, and the isolates were classified into four distinct pathotypes. Four isolates were classified as pathotype P-1 and two as P-4. The remainder did not fit into the existing PSbMV pathotype classification and were tentatively placed into two other groups named U-1 and U-2.

First Report of Mundulla Yellows on Eucalyptus spp. Outside Australia.

Mundulla Yellows (MY) is a newly recognized lethal dieback of unknown etiology affecting Eucalyptus spp. in Australia (1). Progression of symptoms, unlike other disorders described for eucalypts, starts as foliar interveinal chlorosis on one branch followed by branch dieback advancing toward the main trunk. Epicormic shoots with symptomatic leaves typically develop proximal to the dying zone. Continuing dieback leads to tree death in 5 to 20 years. MY trees occur singly or in small foci and do not recover even if affected branches are removed. MY-like symptoms developed on seedlings grafted with bark patches from MY trees (1). There is no evidence for a fungal, phytoplasma, or other bacterial etiology. In a search for a putative virus-like agent we have identified an electrophoretic pattern (2) of several single-stranded RNAs with apparent size of 200 to 600 nt that is consistently associated with MY and absent from disease-free trees (unpublished). The RNAs are not yet characterized, but the pattern can be used as a preliminary confirmatory "fingerprint" for MY. We report here that MY-like symptoms occur in southern Spain and the Spanish island of Ibiza. RNA fingerprints closely resembling those of MY, were detected in symptomatic trees (4 of 4 of E. camaldulensis and 1 of 1 of E. globulus) from Valencia, Spain. MY therefore, appears not to be limited to Australia. References: (1) D. Hanold et al. Landscope 17(4):41, 2002. (2) J. W. Randles. Strategies for implicating virus-like pathogens as the cause of diseases of unknown etiology. Pages 315-332 in: Diagnosis of Plant Virus Diseases, CRC Press, Boca Raton, FL, 1993.

Satellite DNA beta overrides the pathogenicity phenotype of the C4 gene of tomato leaf curl virus but does not compensate for loss of function of the coat protein and V2 genes.

We have investigated the ability of satellite DNA beta to complement mutations in the CP, V2 and C4 genes of the monopartite begomovirus, tomato leaf curl virus, which are potentially involved in movement. A mutation in the coat protein was not complemented by DNA beta. Mutations of the C4 and V2 genes attenuated and abolished symptoms, respectively. In the presence of the C4 mutant, but not the V2 mutant, DNA beta induced typical symptoms, confirming that the satellite encodes a dominant symptom determinant. In contrast to the C4 mutant, DNA beta did not enhance the viral DNA levels of the V2 mutant, suggesting that V2 is required for this phenomenon. The significance of these findings is discussed based on our present understanding of the functions of the viral genes and DNA beta.

Variants of Coconut cadang-cadang viroid isolated from an African oil palm (Elaies guineensis Jacq.) in Malaysia.

Variants of Coconut cadang-cadang viroid have been identified in a plantation oil palm growing in Malaysia. Three size classes are described, comprising 297, 293, and 270 nt. Compared with the 296-nt form of coconut cadang-cadang viroid (CCCVd), all variants substituted C31 --> U in the pathogenicity domain and A175 --> C in the right-hand terminus. Other mutations and deletions accounted for the different sizes. These are the first sequences reported for variants of Coconut cadang-cadang viroid in a species other than coconut palm, and this is the first evidence that variants closely related to CCCVd occur outside the Philippines.

A comparison of the nucleotide sequence homologies of three isolates of bean yellow mosaic virus and their relationship to other potyviruses.

3H-labelled complementary DNA (cDNA) was reverse transcribed from the RNA of three biologically distinct isolates of bean yellow mosaic virus (BYMV-G, Q, and S) by the random primer method of Taylor et al. [Biochim. Biophys. Acta 442, 324-330 (1976)]. Excess virus RNA was hybridized with each of the cDNAs and percentage hybrid formation was assayed using the single-strand-specific nuclease S1, The R0t (1/2) values obtained for the homologous hybridization reactions (1.2 x 10(-2) mol sec liter(-1)) showed that all three BYMV-RNAs were unique species, without detectable zones of reiteration. Reciprocal hybridizations between isolates G, Q, and S have shown that each was significantly different from the other. A comparison of these three isolates with a selection of other potyviruses showed that the relationship between them was closer than that observed between any of them and the other potyviruses tested.

Circularity of the ribonucleic acids associated with cadang-cadang disease.

The ribonucleic acids, ccRNA-1 and ceRNA-2, associated with cadang-cadang disease are circular single-stranded molecules comprising 310 +/- 3 nucleotides and 438 +/- 5 nucleotides, respectively; their molecular weights are estimated to be 1.05 and 1.49 x 10(5) daltons. Therefore ceRNA-2 appears not to be a dimer of ccRNA-1, although it is known to have nucleotide sequences in common with ccRNA-1. Differences in the native structure of the two RNAs as determined by length measurements may account for previously described differences in their properties. Both RNAs resemble viroids, but ccRNA-1 is smaller, and ceRNA-2 is larger, than the viroids which have been characterized.