Multiple approaches to assess the safety of artisanal marine food in a tropical estuary.

In this study, metal and metalloid concentrations and pathogens were measured in shellfish at different locations in a tropical estuary, including sites impacted by sewage and industry. Oyster, mangrove snails and mud snails did not exceed Australian and New Zealand Food Standards maximum levels for copper, lead or estimated inorganic arsenic at any site although copper concentrations in oysters and mud snails exceeded generally expected levels at some locations. Bacterial community composition in shellfish was species-specific regardless of location and different to the surrounding water and sediment. In the snails Telescopium telescopium, Terebralia palustris and Nerita balteata, some bacterial taxa differed between sites, but not in Saccostrea cucullata oysters. The abundance of potential human pathogens was very low and pathogen abundance or diversity was not associated with site classification, i.e. sewage impact, industry impact and reference.

Comparative genome analysis of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A) and "Ca. Phytoplasma asteris" Strains OY-M and AY-WB.

The chromosome sequence of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of "Ca. Phytoplasma australiense" with "Ca. Phytoplasma asteris" strains OY-M and AY-WB showed that the gene order was more conserved between the closely related "Ca. Phytoplasma asteris" strains than to "Ca. Phytoplasma australiense." Differences observed between "Ca. Phytoplasma australiense" and "Ca. Phytoplasma asteris" strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function.

Optimizing Phytoplasma DNA purification for genome analysis.

Genome analysis of uncultivable plant pathogenic phytoplasmas is hindered by the difficulty in obtaining sufficient quantities of phytoplasma enriched DNA. We investigated a combination of conventional enrichment techniques such as cesium chloride (CsCl) buoyant gradient centrifugation, and new methods such as rolling circle amplification (RCA), suppression subtractive hybridization (SSH), and mirror orientation selection (MOS) to obtain DNA with a high phytoplasma:host ratio as the major first step in genome analysis of Candidatus Phytoplasma australiense. The phytoplasma:host ratio was calculated for five different plasmid libraries. Based on sequence data, 90% of clones from CsCl DNA enrichment contained chromosomal phytoplasma DNA, compared to 60% from RCA CsCl DNA and 20% from SSH subtracted libraries. Based on an analysis of representative libraries, none contained plant DNA. A high percentage of clones (80-100%) from SSH libraries contained extrachromosomal DNA (eDNA), and we speculate that eDNA in the original DNA preparation was amplified in subsequent SSH manipulations. Despite the availability of new techniques for nucleic acid amplification, we found that conventional CsCl gradient centrifugation was the best enrichment method for obtaining chromosomal phytoplasma DNA with low host DNA content.

Extrachromosomal DNA isolated from tomato big bud and Candidatus Phytoplasma australiense phytoplasma strains.

The nucleotide sequences of two extrachromosomal elements from tomato big bud (TBB) and one extrachromosomal element from Candidatus Phytoplasma australiense (Ca. P. australiense) phytoplasmas were determined. Both TBB plasmids (3319 and 4092 bp) contained an open reading frame ( approximately 570 bp) with homology to the rolling circle replication initiator protein (Rep). This gene was shorter than the rep genes identified from other phytoplasma plasmids, geminiviruses and bacterial plasmids. Both TBB extrachromosomal DNAs (eDNAs) encoded a putative DNA primase (dnaG) gene, a chromosomal gene required for DNA replication and which contains the conserved topoisomerase/primase domain. We speculate that the replication mechanism for the TBB phytoplasma eDNA involves the dnaG gene instead of the rep gene. The Ca. P. australiense eDNA (3773 bp) was shown to be circular and contained four open reading frames. The rep gene was encoded on ORF 1 and had homology to both plasmid (pLS1) and geminivirus-like domains.

'Candidatus Phytoplasma spartii', 'Candidatus Phytoplasma rhamni' and 'Candidatus Phytoplasma allocasuarinae', respectively associated with spartium witches'-broom, buckthorn witches'-broom and allocasuarina yellows diseases.

Spartium witches'-broom (SpaWB), buckthorn witches'-broom (BWB) and allocasuarina yellows (AlloY) are witches'-broom and yellows diseases of Spartium junceum (Spanish broom), Rhamnus catharticus (buckthorn) and Allocasuarina muelleriana (Slaty she-oak), respectively. These diseases are associated with distinct phytoplasmas. The SpaWB, BWB and AlloY phytoplasmas share <97.5 % 16S rDNA sequence similarity with each other and with other known phytoplasmas, including the closely related phytoplasmas of the apple proliferation group. Also, the SpaWB, BWB and AlloY phytoplasmas each have a different natural plant host. Based on their unique properties, it is proposed to designate the mentioned phytoplasmas as novel 'Candidatus' species under the names 'Candidatus Phytoplasma spartii', 'Candidatus Phytoplasma rhamni' and 'Candidatus Phytoplasma allocasuarinae', respectively.

Detection of Phytoplasmas in Declining Pears in Southern Australia.

Forty-nine pear tree samples collected in Victoria, most of them showing decline symptoms, were tested by polymerase chain reaction (PCR) analysis to detect phytoplasmas. Two universal phytoplasma-specific primer pairs, fP1/rP7 and fU5/rU3, were tested, but only fU5/rU3 amplified the phytoplasma DNA adequately. Nested PCR with universal and group-specific primers, however, proved more effective. Thirty pear trees reacted positively in a nested PCR assay. Restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes MseI and AluI of the PCR fragment amplified with the primer pair fU5/rU3 revealed patterns identical to those from the sweet potato little leaf phytoplasma. This is the first report of a phytoplasma in pear in Australia.

A general method for the detection of potyviral gene products in plant protoplasts and tissue.

A rapid immunostaining procedure for detecting potyviral antigens in individual protoplasts, isolated mesophyll cells, and epidermal strips of Nicotiana tabacum is described. Although all specific antibodies tested detected potyviral antigens in electroporated protoplasts, those against cytoplasmic inclusion (CI) protein provided the most useful results. The number of protoplasts reacting with anti-CI increased with time after inoculation, roughly in parallel with the accumulation of capsid protein, which was measured independently by enzyme-linked immunosorbent assay. Potyviral gene products were also detected in epidermal strips and mesophyll cells separated from systemically infected leaves, indicating that the immunostaining method is generally applicable and that it may prove useful for studying the movement of potyviruses from cell to cell in intact plants.

Isolation of velvet tobacco mottle virus capable of replication with and without a viroid-like RNA.

Field isolates of velvet tobacco mottle virus (VTMoV) induce severe symptoms in Nicotiana clevelandii and encapsidate viroid-like RNA reported to be essential for virus infection. An isolate of the virus producing only mild symptoms on N. clevelandii and devoid of viroid-like RNA has now been isolated from a plant inoculated by a single viruliferous Cyrtopeltis nicotianae, the mirid vector. However, after adding viroid-like RNA isolated from normal VTMoV to the inoculum, the new isolate was shown to support the synthesis of, and encapsidated the viroid-like RNA, thereby reverting to virulence characteristic of the normal isolate. This indicates that the viroid-like RNA can behave as a satellite RNA of VTMoV. The data are discussed in relation to previously published conclusions that viroid-like RNA is essential for the infectivity of VTMoV.