Development of a species-specific recA-based PCR test for Burkholderia fungorum.

The genus Burkholderia comprises over 28 species and species-specific, recA-based polymerase chain reaction (PCR) tests are available for several species, but not for some soil-inhabiting species including B. fungorum. Previous analysis of several novel rhizospheric, environmental isolates belonging to the B. cepacia complex suggested they may be closely related to B. fungorum. To discover any relationship between these isolates and B. fungorum we set out to clone and sequence a portion of the B. fungorum recA gene in order to design species-specific primer pairs for use in a recA-based PCR assay. Using a similar procedure we extended the recA-based PCR assay to identify B. sacchari and B. caledonica, two additional soil-inhabiting Burkholderia spp.

Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus.

A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with 32P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg2+ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3' ends, but different 5' ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.

Molecular characterization of the largest mycoviral-like double-stranded RNAs associated with Amasya cherry disease, a disease of presumed fungal aetiology.

The sequence of the four large (L) double-stranded RNAs (dsRNAs) associated with Amasya cherry disease (ACD), which has a presumed fungal aetiology, is reported. ACD L dsRNAs 1 (5121 bp) and 2 (5047 bp) potentially encode proteins of 1628 and 1620 aa, respectively, that are 37% identical and of unknown function. ACD L dsRNAs 3 (4458 bp) and 4 (4303 bp) potentially encode proteins that are 68% identical and contain the eight motifs conserved in RNA-dependent RNA polymerases (RdRp) of dsRNA mycoviruses, having highest similarity with those of members of the family Totiviridae. Both terminal regions share extensive conservation in all four RNAs, suggesting a functional relationship between them. As ACD L dsRNAs 1 and 2 do not encode RdRps, both are probably replicated by those from either ACD L dsRNA 3 or 4. Partial characterization of the equivalent L dsRNAs 3 and 4 associated with cherry chlorotic rusty spot revealed essentially identical sequences.

Nucleotide sequence and genome organization of Cucurbit yellow stunting disorder virus RNA1.

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA1, a member of the Crinivirus genus in the Closteroviridae, was determined. CYSDV RNA1 is 9126 nucleotides long and contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyl transferase, helicase, and polymerase domains, a small 5 kDa hydrophobic protein and two further downstream ORFs potentially encoding proteins respectively 25 and 22 kDa in size. The genomic position and homology of the four domains comprising the replication module appear to be similar for all sequenced criniviruses but there is divergence in the downstream ORFs, in terms of number, size, position and sequence homology.

Diversity of Burkholderia isolates from woodland rhizosphere environments.

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.

Molecular variation of Potato yellow vein virus isolates.

To evaluate the variation of Potato yellow vein virus from potato fields, 12 isolates were collected from Colombia and one was collected from Peru. Double-stranded RNA was extracted from the plants and used as a template for RT-PCR amplification of the coat protein ( CP) gene and, in separate reactions the C-terminal region of the heat shock protein 70 homologue ( Hsp70h) gene and the N-terminal region of the p60 open reading frame. The CP amplicons were subjected to single-strand conformation polymorphism (SSCP) analysis and, together with the other amplicon, nucleotide sequence analysis. These analyses suggested that there is low genetic diversity in the PYVV isolates examined and that the Peruvian isolate of PYVV may have originated in Colombia.

Analysis of the RNA of Potato yellow vein virus: evidence for a tripartite genome and conserved 3'-terminal structures among members of the genus Crinivirus.

Double-stranded RNA preparations produced from potato plants graft-inoculated with a Peruvian isolate of Potato yellow vein virus (PYVV; genus Crinivirus, family Closteroviridae) contain five RNA species denoted RNA 1, RNA 2, RNA 3, x and y of approximately 8, 5.3, 3.8, 2.0 and 1.8 kbp, respectively. The complete nucleotide sequences of PYVV RNAs 1, 2 and 3 and Northern hybridization analysis showed that PYVV RNA 1 contained the replication module and an additional open reading frame (p7), while two distinct species, RNAs 2 and 3, contain the Closteroviridae hallmark gene array. Pairwise comparisons and phylogeny of genome-encoded proteins showed that PYVV shares significant homology with other criniviruses but is most closely related to the Trialeurodes vaporariorum-vectored Cucumber yellows virus. Secondary structure prediction of the 3'-untranslated regions of all three PYVV RNAs revealed four conserved stem-loop structures and a 3'-terminal pseudoknot structure, also predicted for all fully characterized members of the genus Crinivirus and some members of the genera Closterovirus and Ampelovirus.