Growth of V. parahaemolyticus in Tropical Blacklip Rock Oysters.

The opportunistic pathogen Vibrio parahaemolyticus poses a significant food safety risk worldwide, and understanding its growth in commercially cultivated oysters, especially at temperatures likely to be encountered post-harvest, provides essential information to provide the safe supply of oysters. The Blacklip Rock Oyster (BRO) is an emerging commercial species in tropical northern Australia and as a warm water species, it is potentially exposed to Vibrio spp. In order to determine the growth characteristics of Vibrio parahaemolyticus in BRO post-harvest, four V. parahaemolyticus strains isolated from oysters were injected into BROs and the level of V. parahaemolyticus was measured at different time points in oysters stored at four temperatures. Estimated growth rates were -0.001, 0.003, 0.032, and 0.047 log10 CFU/h at 4 °C, 13 °C, 18 °C, and 25 °C, respectively. The highest maximum population density of 5.31 log10 CFU/g was achieved at 18 °C after 116 h. There was no growth of V. parahaemolyticus at 4 °C, slow growth at 13 °C, but notably, growth occurred at 18 °C and 25 °C. Vibrio parahaemolyticus growth at 18 °C and 25 °C was not significantly different from each other but were significantly higher than at 13 °C (polynomial GLM model, interaction terms between time and temperature groups p < 0.05). Results support the safe storage of BROs at both 4 °C and 13 °C. This V. parahaemolyticus growth data will inform regulators and assist the Australian oyster industry to develop guidelines for BRO storage and transport to maximise product quality and safety.

Sources and drivers of contamination along an urban tropical river (Ciliwung, Indonesia): Insights from microbial DNA, isotopes and water chemistry.

Wastewater treatment infrastructure is lacking in many developing countries, often resulting in high loads of contaminants discharged to urban rivers. In these countries, targeted pollution mitigation requires an understanding of where, how and when contaminants enter water bodies. Here we report on contamination of the Ciliwung River, a dynamic, tropical system flowing through the Jakarta metropolitan area (Indonesia). We measured a set of isotopic, chemical and microbial tracers in representative water and contamination sources, as well as longitudinally within the river, to assess the spatial and temporal variations in contaminant levels in and pathways to the river. In the dry season, we observed a tight coupling between locally recharged groundwater sources and the river, whereas in the wet season, one single water source originating from the fractured headwaters predominantly contributed to river flow. Yet, the flushing of upstream waters in the wet season did not always lead to the dilution of contaminants downstream. We delineated several contamination hotspots along the river, particularly active during the wet season due to higher hydrological connectivity between sources and the river. These hotspots may originate from septic tank leakage, as supported by metal ratios and dominant microbial communities, although we could not rule out other potential sources such as urban runoff or sediment resuspension. Bayesian source tracking on the whole microbial community proved useful in outlining processes that conventional tracers did not capture, such as the occurrence of a localised domestic contamination in the upper catchment, and the inflow of agricultural runoff all along the river profile during the wet season. Our study emphasises the role of rivers as biogeochemical reactors that constantly process and transform contaminants and microbial communities. We also demonstrate the value of using isotopic, chemical and microbial tools together to trace the movement of water and contaminants through urban rivers.

Soil uranium concentration at Ranger Uranium Mine Land Application Areas drives changes in the bacterial community.

Soil microorganisms may respond to metal stress by a shift in the microbial community from metal sensitive to metal resistant microorganisms. We assessed the bacterial community from low (2-20 mg kg-1), medium (200-400 mg kg-1), high (500-900 mg kg-1) and very high (>900 mg kg-1) uranium soils at Ranger Uranium Mine in northern Australia through pyrosequencing. Proteobacteria (28.85%) was the most abundant phylum at these sites, followed by Actinobacteria (9.31%), Acidobacteria (7.33%), Verrucomicrobia (2.11%), Firmicutes (2.02%), Chloroflexi (1.11%), Cyanobacteria (0.93%), Planctomycetes (0.82%), Bacteroidetes (0.46%) and Candidate_division_WS3 (Latescibacteria) (0.21%). However, 46.79% of bacteria were unclassified. Bacteria at low U soils differed from soils with elevated uranium. Bacterial OTUs closely related to Kitasatospora spp., Sphingobacteria spp. and Rhodobium spp. were only present at higher uranium concentrations and the bacterial community also changed with seasonal and temporal changes in soil uranium and physicochemical variables. This study using next generation sequencing in association with environmental variables at a uranium mine has laid a foundation for further studies of soil-microbe-metal interactions which may be useful for developing sustainable management and rehabilitation strategies. Furthermore, bacterial species associated with higher uranium may serve as useful indicators of uranium contamination in the wet-dry tropics.

Differences in structure of northern Australian hypolithic communities according to location, rock type, and gross morphology.

Hypolithic communities (under translucent rocks) were compared between a semi-arid site (Wave Hill) and a site with considerably higher rainfall (Lake Bennett) to test the hypothesis that the communities at the higher rainfall site would be more diverse. A total of 153 cyanobacteria operational taxonomic units (OTUs) were identified, and only 50 of those were found at both sites. Of these, only two were core OTUs, as defined as being present in ≥90% of samples, highlighting the extreme differences in the cyanobacterial communities at the two sites. At Wave Hill, we compared the composition of the cyanobacterial components under two different rock types (quartz and prehnite) to determine if the different minerals would result in different hypolithic communities, but no differences were found. Of the 42 core OTUs found at Wave Hill, 22 (52%) were shared between the two rock types. As hypothesised, the diversity of both cyanobacteria and eukaryotes in the hypolithic communities was significantly higher at Lake Bennett. Some hypolithic communities were thin and tightly adhered to the rock surface, but others were thicker and could be peeled off the rock in sheets. However, the two types were not significantly different in OTU composition. Metazoans, primarily nematodes, were ubiquitous, raising the possibility that nematodes may act as vectors to transport the components of hypolithic communities from rock to rock as a mechanism of colonization.

Cadmium uptake and zinc-cadmium antagonism in Australian tropical rock oysters: Potential solutions for oyster aquaculture enterprises.

Variable and occasionally high concentrations of cadmium in wild oysters at a remote location with the potential to develop aquaculture enterprises motivated research into the distribution and sources of metals in oysters, seawater, sediment, suspended solids and phytoplankton. Saccostrea mytiloides and Saccostrea mordax contained cadmium concentrations exceeding the food standard maximum level (ML) at three of four sites. At one site with high zinc levels in sediment, oyster cadmium levels were below the ML. Metal levels in seawater were not correlated with cadmium levels in oysters but high cadmium/zinc ratios were measured in Trichodesmium erythraeum blooms. We suggest that oysters accumulate cadmium mainly from annual phytoplankton blooms except at sites where zinc availability is sufficiently high to prevent uptake though a mechanism of antagonistic exclusion. Knowledge of the source and uptake mechanisms of cadmium in oysters should lead to new management strategies to reduce cadmium levels in farmed oysters.

Enrichment of 15N/14N in wastewater-derived effluent varies with operational performance of treatment systems: implications for isotope monitoring in receiving environments.

Stable nitrogen isotope ratios are routinely used to trace the dispersion and assimilation of wastewater-derived N in receiving environments, but few isotope studies have investigated wastewater treatment plants and ponds themselves. An improved understanding of N isotope compositions in effluent will help assess treatment plant processes and performance and will help trace sources of excess nutrients in receiving environments. Here, we assess N budgets and treatment processes in seven wastewater treatment plants and wastewater stabilisation ponds in northern Australia based on concentrations and isotope ratios of N in effluent. We show that δ15N values in effluent are linked to treatment type, effectiveness of conversion of ammonia and levels of gaseous N emissions. These relationships suggest that N isotope monitoring of wastewater treatment plants and ponds can provide an integrated assessment of treatment performance and gaseous N emissions on a pond- or plant-wide scale that is not readily available through other methods. Our findings further imply that monitoring N isotope ratios in receiving environments cannot be assumed to be universally effective as their sensitivity to uptake of wastewater-derived N will vary with the characteristics of individual treatment systems. Paradoxically, N isotope monitoring is less effective where treatment systems are functioning poorly and where monitoring needs are the greatest.

Land application of mine water causes minimal uranium loss offsite in the wet-dry tropics: Ranger Uranium Mine, Northern Territory, Australia.

Ranger Uranium Mine (RUM) is situated in the wet-dry tropics of Northern Australia. Land application (irrigation) of stockpile (ore and waste) runoff water to natural woodland on the mine lease is a key part of water management at the mine. Consequently, the soil in these Land Application Areas (LAAs) presents a range of uranium (U) and other metals concentrations. Knowledge of seasonal and temporal changes in soil U and physicochemical parameters at RUM LAAs is important to develop suitable management and rehabilitation strategies. Therefore, soil samples were collected from low, medium, high and very high U sites at RUM LAAs for two consecutive years and the effect of time and season on soil physicochemical parameters particularly U and other major solutes applied in irrigation water was measured. Concentrations of some of the solutes applied in the irrigation water such as sulphur (S), iron (Fe) and calcium (Ca) showed significant seasonal and temporal changes. Soil S, Fe and Ca concentration decreased from year 1 to year 2 and from dry to wet seasons during both years. Soil U followed the same pattern except that we recorded an increase in soil U concentrations at most of the RUM LAAs after year 2 wet season compared to year 2 dry season. Thus, these sites did not show a considerable decrease in soil U concentration from year 1 to year 2. Sites which contained elevated U after wet season 2 also had higher moisture content which suggests that pooling of U containing rainwater at these sites may be responsible for elevated U. Thus, U may be redistributed within RUM LAAs due to surface water movement. The study also suggested that a decrease in U concentrations in LAA soils at very high U (>900 mg kg(-1)) sites is most likely due to transport of particulate matter bound U by surface runoff and U may not be lost from the surface soil due to vertical movement through the soil profile. Uranium attached to particulate matter may reduce its potential for environmental impact. These findings suggest that U is effectively adsorbed by the soils and thus land application may serve as a useful tool for U management in the wet-dry tropics of northern Australia.

Saltwater intrusion history shapes the response of bacterial communities upon rehydration.

Saltwater intrusion (SWI) can result in the loss of dominant vegetation from freshwater habitats. In northern Australia, sea level is predicted to rise 17-50 cm by 2030-2070. This will exacerbate the impact of SWI, threatening Ramsar-listed habitats. Soil bacteria in these habitats play a significant role in biogeochemical cycling, regulating availability of essential nutrients such as nitrogen to vegetation. However, there is limited understanding as to how SWI will impact these soil bacteria. Floodplain soil samples were collected from the South Alligator River floodplain in Northern Australia from sites with contrasting histories of SWI. A SWI event was simulated over 7 days with treatments of saltwater and freshwater. Bacterial community composition before and after treatment were measured using next generation sequencing of bacterial DNA. Sites with no history of SWI showed no significant changes in community taxonomic composition following treatments, suggesting the community at these sites have broad functional capacity which may be due to their historic conditioning over many years. Sites with a history of SWI showed a significant response to both treatments. Following saltwater treatment, there was an increase in sulfate-reducing bacteria, which are known to have an impact on carbon and nitrogen cycling. We suggest that the impact of SWI causes a shift in the soil bacteria which alters the community to one which is more specialised, with implications for the cycling of essential elements and nutrients.

The diversity and abundance of polychaetes (Annelida) are altered in sediments impacted by alumina refinery discharge in the Northern Territory, Australia.

We collected polychaete diversity and abundance data at a range of impacted and reference sites near an alumina refinery in Melville Bay, northern Australia. The aims were to measure the impact of sediment modified by the alumina refinery discharge on polychaete communities and secondly to gather baseline data from which to measure future changes. Polychaete communities in both soft-bottom habitats and subtidal areas adjacent to mangrove forests were studied. We also developed and deployed an artificial substratum device to sample polychaetes associated with hard-substrate habitats. For each habitat, polychaete community composition was different between impacted and reference sites and at multiple time points. The impact of future changes either from bioremediation or management practices can be measured against these baseline data. Indicator species analysis was used to identify polychaete species that were significantly different at the locations tested, and we discuss their potential as indicator species.

Fungi outcompete bacteria under increased uranium concentration in culture media.

As a key part of water management at the Ranger Uranium Mine (Northern Territory, Australia), stockpile (ore and waste) runoff water was applied to natural woodland on the mine lease in accordance with regulatory requirements. Consequently, the soil in these Land Application Areas (LAAs) presents a range of uranium concentrations. Soil samples were collected from LAAs with different concentrations of uranium and extracts were plated onto LB media containing no (0 ppm), low (3 ppm), medium (250 ppm), high (600 ppm) and very high (1500 ppm) uranium concentrations. These concentrations were similar to the range of measured uranium concentrations in the LAAs soils. Bacteria grew on all plates except for the very high uranium concentrations, where only fungi were recovered. Identifications based on bacterial 16S rRNA sequence analysis showed that the dominant cultivable bacteria belonged to the genus Bacillus. Members of the genera Paenibacillus, Lysinibacillus, Klebsiella, Microbacterium and Chryseobacterium were also isolated from the LAAs soil samples. Fungi were identified by sequence analysis of the intergenic spacer region, and members of the genera Aspergillus, Cryptococcus, Penicillium and Curvularia were dominant on plates with very high uranium concentrations. Members of the Paecilomyces and Alternaria were also present but in lower numbers. These findings indicate that fungi can tolerate very high concentrations of uranium and are more resistant than bacteria. Bacteria and fungi isolated at the Ranger LAAs from soils with high concentrations of uranium may have uranium binding capability and hence the potential for uranium bioremediation.

The chemical composition and bacteria communities in acid and metalliferous drainage from the wet-dry tropics are dependent on season.

Acid and metalliferous drainage (AMD) occurs when sulphidic minerals, such as arsenopyrite, chalcopyrite and pyrite, are exposed to oxygen and water. Climate, geology and mine site practices can have a significant impact on AMD composition. The elemental composition of the AMD can also affect the bacterial community. Our hypothesis was that in the dry season the AMD at two mine sites, Rum Jungle and Mt Todd, in the Northern Territory, Australia, has a higher concentration of dissolved metals because standing water evaporates during the extended dry period. Our second hypothesis was that the wet and dry season bacteria community in AMD at Rum Jungle and Mt Todd are different, and this difference is correlated to seasonally specific changes in physicochemistry. The first hypothesis was tested by measuring elemental concentrations in AMD during the wet and dry seasons at Mt Todd and Rum Jungle mine sites. The physicochemical properties such as temperature, pH and dissolved oxygen were also measured. To test the second hypothesis, we extracted DNA from AMD samples collected at Rum Jungle and Mt Todd during the wet and dry seasons. The hypervariable V6 region of the bacterial 16S rRNA gene was sequenced by 454 pyrosequencing. The bacterial community composition was examined and related to physiochemical variables. The elemental concentrations in Rum Jungle AMD were higher in the dry season compared to the wet season, but at Mt Todd the elemental composition of AMD changed with year, rather than season. The bacteria community in AMD at Rum Jungle changed between the wet and dry season while in Mt Todd AMD the bacteria community from year 1 was significantly different from year 2. The data showed that the elemental composition and bacteria communities of AMD at Rum Jungle and Mt Todd are influenced by season, mine site practices and geological characteristics of the ore body. In addition, the iron oxidising bacteria Leptospirillum and Acidithiobacillus typically associated with AMD in temperate regions were not prevalent at out tropical study sites.

The transcriptome and proteome are altered in marine polychaetes (Annelida) exposed to elevated metal levels.

Polychaetes are often used in toxicological studies to understand mechanisms of resistance and for biomarker detection, however, we know of only a few genetic pathways involved in resistance. We found the marine polychaete Ophelina sp.1 (Opheliidae) in sediment containing high copper levels and investigated this phenomenon by measuring metal accumulation in the worms and changes in gene and protein expression. We sequenced the transcriptome of Ophelina sp.1 from both the impacted and reference sediments using 454-sequencing and analysed their proteomes using differential in gel electrophoresis (DIGE). We used the sequenced transcriptome to guide protein identification. Transcripts coding for the copper chaperone, Atox1, were up-regulated in the worms inhabiting the high copper sediment. In addition, genes coding for respiratory proteins, detoxification proteins and cytoskeletal proteins were significantly altered in metal-exposed worms; many of these changes were also detected in the proteome. This dual approach has provided a better understanding of heavy metal resistance in polychaetes and we now have a wider range of suitable indicator genes and proteins for future biomarker development.

The bacterial community associated with the marine polychaete Ophelina sp.1 (Annelida: Opheliidae) is altered by copper and zinc contamination in sediments.

Tolerant species of polychaete worms can survive in polluted environments using various resistance mechanisms. One aspect of resistance not often studied in polychaetes is their association with symbiotic bacteria, some of which have resistance to metals and may help the organism to survive. We used "next generation" 454 sequencing of bacterial 16S rRNA sequences associated with polychaetes from a copper- and zinc-polluted harbor and from a reference site to determine bacterial community structure. We found changes in the bacteria at the polluted site, including increases in the abundance of bacteria from the order Alteromonadales. These changes in the bacteria associated with polychaetes may be relatively easy to detect and could be a useful indicator of metal pollution.

Microclimate and limits to photosynthesis in a diverse community of hypolithic cyanobacteria in northern Australia.

Hypolithic microbes, primarily cyanobacteria, inhabit the highly specialized microhabitats under translucent rocks in extreme environments. Here we report findings from hypolithic cyanobacteria found under three types of translucent rocks (quartz, prehnite, agate) in a semiarid region of tropical Australia. We investigated the photosynthetic responses of the cyanobacterial communities to light, temperature and moisture in the laboratory, and we measured the microclimatic variables of temperature and soil moisture under rocks in the field over an annual cycle. We also used molecular techniques to explore the diversity of hypolithic cyanobacteria in this community and their phylogenetic relationships within the context of hypolithic cyanobacteria from other continents. Based on the laboratory experiments, photosynthetic activity required a minimum soil moisture of 15% (by mass). Peak photosynthetic activity occurred between approximately 8 degrees C and 42 degrees C, though some photosynthesis occurred between -1 degrees C and 51 degrees C. Maximum photosynthesis rates also occurred at light levels of approximately 150-550 micromol m(-2) s(-1). We used the field microclimatic data in conjunction with these measurements of photosynthetic efficiency to estimate the amount of time the hypolithic cyanobacteria could be photosynthetically active in the field. Based on these data, we estimated that conditions were appropriate for photosynthetic activity for approximately 942 h (approximately 75 days) during the year. The hypolithic cyanobacteria community under quartz, prehnite and agate rocks was quite diverse both within and between rock types. We identified 115 operational taxonomic units (OTUs), with each rock hosting 8-24 OTUs. A third of the cyanobacteria OTUs from northern Australia grouped with Chroococcidiopsis, a genus that has been identified from hypolithic and endolithic communities from the Gobi, Mojave, Atacama and Antarctic deserts. Several OTUs identified from northern Australia have not been reported to be associated with hypolithic communities previously.

Use of Survival Analysis to Determine the Postincubation Time-to-Death of Papaya Due to Yellow Crinkle Disease in Australia.

The current management recommendation for papaya (Carica papaya) plants exhibiting symptoms of yellow crinkle disease in Australia is the practice of ratooning infected plants. Ratooning involves removing the main stem of diseased papaya plants and allowing a lateral stem (supposedly pathogen-free) to develop and replace the diseased stem. Using nonparametric and parametric methods of survival analysis, we tested different hypotheses regarding plant factors that may influence the postincubation period survival time of phytoplasma-infected papaya. The factors included plant age, the season (wet versus dry) when papaya plants first became symptomatic, and the two predominant phytoplasma strains causing papaya yellow crinkle: tomato big bud (TBB) or sweet potato little leaf strain V4 (SPLL-V4). Median survival time was estimated to be from 4 to 5 months. Therefore, we estimated that the infectious period (incubation period plus the period from postincubation to time-to-death period) of infected papaya ranges from 6 to 9 months. Using parametric accelerated failure modeling and nonparametric Cox proportional hazard modeling, no significant improvement from a null model (no covariates) was found when analyzing plant age, the season a plant was observed to be symptomatic, or phytoplasma strain. However, the season in which a papaya plant became symptomatic differed between the two phytoplasma strains, indicating that the TBB and SPLL-V4 strains may have different modes of insect acquisition and transmission. Because of the long infectious period and the rate of plantto-plant spread, we question the use of ratooning as the primary management tactic for managing papaya yellow crinkle.

Identification of genes in the tomato big bud phytoplasma and comparison to those in sweet potato little leaf-V4 phytoplasma.

Genetic relatedness of phytoplasmas is commonly defined on the basis of differences in the highly conserved 16S rRNA gene, which may not resolve closely related phytoplasmas. An example of this is the closely related tomato big bud (TBB) and sweet potato little leaf strain V4 (SPLL-V4) phytoplasmas, which cannot easily be differentiated by their 16S rRNA gene sequences. This study aimed to identify genes on the TBB phytoplasma chromosome which could be used to examine genetic variation between these two closely related phytoplasmas. Random clones generated from TBB phytoplasma genomic DNA were sequenced and characterized by database analysis. Twenty-three genes were identified within 19 random clones, which contained approximately 18.0 kbp of TBB phytoplasma genomic DNA. Half of the TBB phytoplasma genes identified were involved in DNA replication, transcription and translation. The remaining TBB phytoplasma genes were involved in protein secretion, cellular processes and energy metabolism. Phylogenetic analysis of representative genes showed that the TBB phytoplasma grouped with the mycoplasmas with the exception of the TBB phytoplasma secA gene, which grouped with the onion yellows phytoplasma. PCR primers were designed based on the new genes and tested on isolates of the TBB and SPLL-V4 phytoplasmas. Most primers amplified a product from TBB and SPLL-V4 phytoplasma samples. When amplified products were subjected to RFLP analysis, the restriction patterns were the same as the respective original clones. This result confirmed that the same sequence had been amplified by PCR and showed that these isolates were indistinguishable using the new genes. This study showed that in fact the TBB and SPLL-V4 phytoplasmas are closely related even with the analysis of new genes. These new genes have, however, provided insight into the biology of the TBB and SPLL-V4 phytoplasmas.

Chromosome mapping of the sweet potato little leaf phytoplasma reveals genome heterogeneity within the phytoplasmas.

To further understand the genomic diversity and genetic architecture of phytoplasmas, a physical and genetic map of the sweet potato little leaf (SPLL) strain V4 phytoplasma chromosome was determined. PFGE was used to determine the size of the SPLL-V4 genome, which was estimated to be 622 kb. A physical map was prepared by two-dimensional reciprocal digestions using the restriction endonucleases BssHII, Smal, Eagl and I-Ceul. Sixteen cleavage sites were located on the map. Southern hybridizations of digested SPLL-V4 chromosomal DNA were done using random clones and PCR-amplified genes as probes. This confirmed fragment positions and located the two rRNA operons and the linked fus/tuf genes encoding elongation factors G and Tu, respectively, on the physical map. An inversion of one of the rRNA operons was observed from hybridization data. Sequence analysis of one of the random clones identified a gid gene encoding a glucose-inhibited division protein. Digestions of the tomato big bud (TBB) phytoplasma chromosome with the same four enzymes revealed genome heterogeneity when compared to the closely related SPLL-V4, and a preliminary chromosome size for the TBB phytoplasma of 662 kb was estimated. This mapping information has revealed that significant genome diversity exists within the phytoplasmas.

Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas.

Primers designed from sequences of the gene encoding the elongation factor Tu (tuf gene) of several culturable mollicutes amplified most of the tuf gene from phytoplasmas of the aster yellows, stolbur and X-disease groups. About 85% of the tuf gene from two aster yellows strains and a tomato stolbur phytoplasma was sequenced. The nucleotide sequence similarity between these related phytoplasmas was between 87.8 and 97.0%, whereas the homology with other mollicutes was 66.3-72.7%. The similarity of the deduced amino acid sequence was significantly higher, ranging from 96.0 to 99.4% among the phytoplasmas and 78.5% to 83.3% between phytoplasmas and the culturable mollicutes examined. From the nucleotide sequences of the phytoplasma strains, two pairs of primers were designed; one amplified the phytoplasmas of most phylogenetic groups that were established, the other was specific for the aster yellows and stolbur groups. The phytoplasmas of the various groups that were amplified could be distinguished by RFLP analysis using Sau3AI, Alul and HpaII. The aster yellows group could be divided into five Sau3AI RFLP groups. These results showed that the tuf gene has the potential to be used to differentiate and classify phytoplasmas. Southern blot analysis revealed that the tuf gene is present as a single copy.