Anna C. Padovan

Detection and differentiation of phytoplasmas in Australia: an update

Phytoplasmas were found in 33 plant species that were not described as host plants in an earlier Australian survey. Plants displayed characteristic symptoms of little leaf, proliferation, and floral abnormalities. Restriction fragment length polymorphism analysis revealed 13 different restriction patterns. The majority of phytoplasmas showed a restriction pattern identical to that of either the tomato big bud (TBB) or sweet potato little leaf V4 (SPLL-V4) phytoplasma. Phytoplasmas from 6 plant species showed a restriction pattern similar to that of the pigeonpea little leaf (PLL) phytoplasma. One phytoplasma from garden bean displayed a restriction pattern identical to that found in papaya dieback and Australian grapevine yellows (AGY) phytoplasmas. Seven new restriction fragment patterns have been detected and sequence analysis of the 16S/23S spacer region revealed that 3 of these phytoplasmas are related to the faba bean phyllody (FBP) group. The spacer region of a graminaceous phytoplasma was most similar to phytoplasmas from the sugarcane white leaf group. Another graminaceous phytoplasma was identical to a phytoplasma from Indonesia. The spacer region of a phytoplasma from poinsettia (PoiBI) was identical to the western X-disease phytoplasma from North America and Europe. The spacer region of a phytoplasma in stylosanthes contained no tRNAIle. Full-length 16S rRNA gene sequences from selected new phytoplasmas were determined to corroborate results obtained from the spacer region analyses. Three of these phytoplasmas (galactia little leaf, vigna little leaf, and stylosanthes little leaf) are, along with the PoiBI phytoplasma and the graminaceous phytoplasmas, members of phytoplasma groups that have not been reported before in Australia.

'Candidatus Phytoplasma australiense' is the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases

Sequence comparisons and phylogenetic analysis of the 16S rRNA genes and the 16S/23S spacer regions of the phytoplasmas associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases revealed minimal nucleotide differences between them resulting in the formation of a monophyletic group. Therefore, along with Australian grapevine yellows, the phytoplasmas associated with Phormium yellow leaf and papaya dieback should also be considered as ‘Candidatus Phytoplasma australiense’.

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.

Multiple approaches to microbial source tracking in tropical northern Australia

Microbial source tracking is an area of research in which multiple approaches are used to identify the sources of elevated bacterial concentrations in recreational lakes and beaches. At our study location in Darwin, northern Australia, water quality in the harbor is generally good, however dry‐season beach closures due to elevated Escherichia coli and enterococci counts are a cause for concern. The sources of these high bacteria counts are currently unknown. To address this, we sampled sewage outfalls, other potential inputs, such as urban rivers and drains, and surrounding beaches, and used genetic fingerprints from E. coli and enterococci communities, fecal markers and 454 pyrosequencing to track contamination sources. A sewage effluent outfall (Larrakeyah discharge) was a source of bacteria, including fecal bacteria that impacted nearby beaches. Two other treated effluent discharges did not appear to influence sites other than those directly adjacent. Several beaches contained fecal indicator bacteria that likely originated from urban rivers and creeks within the catchment. Generally, connectivity between the sites was observed within distinct geographical locations and it appeared that most of the bacterial contamination on Darwin beaches was confined to local sources.

Weed hosts ofFusarium oxysporum f. sp.cubense tropical race 4 in northern Australia

Roots from 18 plant species collected from two banana plantation sites infested withFusarium oxysporum f. sp.cubense (Foe) tropical race 4 in northern Australia were analysed for the presence of the banana pathogen. Foe tropical race 4 (VCG 01213/16) was isolated from one monocotyledenous speciesChloris inflata and three dicotolydenous species,Euphorbia heterophylla, Tridax procumbens and Cyanthilium cinereum. All four species are common in disturbed areas throughout northern Australia. Current management protocols for fusarium wilt of banana involve the immediate destruction of symptomatic banana plants to reduce fungal sporulation, but do not consider alternative weed hosts. If root colonisation of non-host species byFoc tropical race 4 enables the pathogen to persist, even in fallow fields, weed management must be considered when managing this disease.

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.

Phytoplasmas associated with diseases in strawberry

Strawberry plants with green petal and lethal yellows diseases collected in south Queensland were infected with the same phytoplasma that causes dieback of papaya.

A rod-like bacterium is responsible for high molybdenum concentrations in the tropical sponge Halichondria phakellioides

The tropical marine sponge, Halichondria phakellioides, from Darwin Harbour contains high concentrations of molybdenum. A rod-like bacterium extracellular in sponge tissue was observed using transmission electron microscopy. Molybdenum was located within these bacteria, but not in sponge cells. This is the first report of the trace element molybdenum localised in a sponge bacterial symbiont. Many different bacterial symbionts were identified in the sponge by sequence analysis so the identity of the molybdenum-accumulating bacterium could only be inferred.

Microbial signatures can help distinguish moon sponges (family Tetillidae) from Darwin Harbour, Australia

The bacterial communities of two sponge morphs collected as part of an ecological study and initially allocated to the genus Paratetilla (Demospongiae: Spirophorida: Tetillidae) were analysed using denaturing gradient gel electrophoresis (DGGE) targeting a region of the 16S rRNA gene. The results showed that the two morphs had different bacterial communities, which suggested that they might be distinct Paratetilla species. The sponge samples were further analysed using conventional taxonomy and cytochrome oxidase I (COI) gene sequencing. These data confirmed that (1) the two morphs belonged to different species, and (2) one morph was more closely related to the tetillid genus Cinachyrella than to Paratetilla.

Acinetobacter baumannii detected on mCCDA medium in a waste stabilisation pond

Acinetobacter baumannii survives for prolonged periods under a wide range of environmental conditions. In a larger study investigating the efficacy of pathogen removal in a waste stabilization ponds (WSP), we cultivated microbes from wastewater samples on mCCDA agar containing selective and recommended supplements for the growth of Campylobacter. This bacterium is a recommended reference pathogen for the verification and validation of water recycling schemes in Australia and other parts of the world. A high number of colonies characteristic of Campylobacter grew on the selective media but this did not correlate with qPCR data. Using primers targeting the16S rRNA gene, and additional confirmatory tests such as detection of VS1, ompA, blaOXA-51-like, blaOXA-23-like genes, we tested eight random colonies from eight samples (64 colonies in total) and identified them as A. baumannii. Wastewater grab samples taken three times over 6 months throughout the WSP system showed removal of A. baumannii in the WSP atrates similar to E. coli. In contrast, further intensive sampling from the inlet and the outlet of the WSP using a refrigerated auto-sampler showed that the number of A. baumannii in most sampling rounds did not differ significantly between the inlet and outlet of the WSP and that there was high variation between replicates at the outlet only. Resistance genes were detected in most A. baumannii isolated from the waste stabilisation pond and may potentially be a source of antibiotic resistance for environmental strains.