Aspects of the interaction between Xanthorrhoea australis and Phytophthora cinnamomi in south-western Victoria, Australia

Diseases in natural ecosystems are often assumed to be less severe than those observed in domestic cropping systems due to the extensive biodiversity exhibited in wild vegetation communities. In Australia, it is this natural biodiversity that is now under threat from Phytophthora cinnamomi. The soilborne Oomycete causes severe decline of native vegetation communities in south-western Victoria, Australia, disrupting the ecological balance of native forest and heathland communities. While the effect of disease caused by P. cinnamomi on native vegetation communities in Victoria has been extensively investigated, little work has focused on the Anglesea healthlands in south-western Victoria. Nothing is known about the population structure of P. cinnamomi at Anglesea. This project was divided into two main components to investigate fundamental issues affecting the management of P. cinnamomi in the Anglesea heathlands. The first component examined the phenotypic characteristics of P. cinnamomi isolates sampled from the population at Anglesea, and compared these with isolates from other regions in Victoria, and also from Western Australia. The second component of the project investigated the effect of the fungicide phosphonate on the host response following infection by P. cinnamomi. Following soil sampling in the Anglesea heathlands, a collection of P, cinnamomi isolates was established. Morphological and physiological traits of each isolate were examined. All isolates were found to be of the A2 mating type. Variation was demonstrated among isolates in the following characteristics: radial growth rate on various nutrient media, sporangial production, and sporangial dimensions. Oogonial dimensions did not differ significantly between isolates. Morphological and physiological variation was rarely dependant on isolate origin. To examine the genetic diversity among isolates and to determine whether phenotypic variation observed was genetically based, Random Amplified Polymorphic DNA (RAPD) analyses were conducted. No significant variation was observed among isolates based on an analysis of molecular variance (AMQVA). The results are discussed in relation to population biology, and the effect of genetic variation on population structure and population dynamics. X australis, an arborescent monocotyledon indigenous to Australia, is highly susceptible to infection by P. cinnamomi. It forms an important component of the heathland vegetation community, providing habitat for native flora and fauna, A cell suspension culture system was developed to investigate the effect of the fungicide phosphonate on the host-pathogen interaction between X. australis and P. cinnamomi. This allowed the interaction between the host and the pathogen to be examined at a cellular level. Subsequently, histological studies using X. australis seedlings were undertaken to support the cellular study. Observations in the cell culture system correlated well with those in the plant. The anatomical structure of X australis roots was examined to assist in the interpretation of results of histopathological studies. The infection of single cells and roots of X. australis, and the effect of phosphonate on the interaction are described. Phosphonate application prior to inoculation with P. cinnamomi reduced the infection of cells in culture and of cells in planta. In particular, phosphonate was found to stimulate the production of phenolic material in roots of X australis seedlings and in cells in suspension cultures. In phosphonate-treated roots of X australis seedlings, the deposition of electron dense material, possibly lignin or cellulose, was observed following infection with P. cinnamomi. It is proposed that this is a significant consequence of the stimulation of plant defence pathways by the fungicide. Results of the study are discussed in terms of the implications of the findings on management of the Anglesea heathlands in Victoria, taking into account variation in pathogen morphology, pathogenicity and genotype. The mode of action of phosphonate in the plant is discussed in relation to plant physiology and biochemistry.

Molecular depth profiling of organic and biological materials

Atomic depth profiling using secondary ion mass spectrometry, SIMS, is common in the field micro-electronics; however, the generation of molecular information as a function of sample depth is difficult due to the accumulation of damage both on and beneath the sample surface. The introduction of polyatomic ion beams such as SF₅ and C₆₀ have raised the possibility of overcoming this problem as they deposit the majority of their energy in the upper surface of the sample resulting in increased sputter yields but with a complimentary reduction in sub-surface damage accumulation. In this paper we report the depth profile analysis of the bio-polymer polycaprolactone, PCL, using the polyatomic ions Au₃⁺ and C₆₀⁺ and the monoatomic Au+. Results are compared to recent analysis of a similar sample using . depth profiling of cellulose is also demonstrated, an experiment that has been reported as unsuccessful when attempted with implications for biological analysis are discussed.

The last piece in the cellulase puzzle : the characterisation of β-glucosidase from the herbivorous gecarcinid land crab Gecarcoidea natalis

A 160 kDa enzyme with β-glucosidase activity was purified from the midgut Gland of the land crab Gecarcoidea natalis. The enzyme was capable of releasing glucose progressively from cellobiose, cellotriose or cellotetraose. Although β-glucosidases (EC 3.2.1.21) have some activity towards substrates longer than cellobiose, the enzyme was classified as a glucohydrolase (EC 3.2.1.74) as it had a preference for larger substrates (cellobiose<cellotriose=cellotetraose). It was able to synthesise some cellotetraose by the transglycosylation of smaller substrates – another common feature of glucohydrolases. The interaction between the glucohydrolase described here and the endo-β-1,4-glucanases described previously for G. natalis provides a complete model for cellulose hydrolysis in crustaceans and possibly in other invertebrates. After mechanical fragmentation by the gastric mill, multiple endo-β-1,4-glucanases would initially cleave β-1,4-glycosidic bonds within native cellulose, releasing small oligomers, including cellobiose, cellotriose and cellotetraose. The glucohydrolase would then attach to these oligomers, progressively releasing glucose. The glucohydrolase might also attach directly to crystalline cellulose to release glucose from free chain ends. This two-enzyme system differs from the traditional model, which suggests that total cellulose hydrolysis requires the presence an endo-β-1,4-glucanse, a cellobiohydrolase and a β-glucosidase

Tailoring enzymes for biofuel production with reference to Australian biomass

Manufacture of biofuels from existing biomass may provide a sustainable alternative to the extensive utilization of fossil fuels. Biomass offers environmental advantage over fossil fuels as it is a renewable energy source with low sulphur and nitrogen content and is carbon neutral over its production and utilization. Ranges of biomass are reported worldwide to be suitable raw material for bioethanol production. These can be generally classified into three groups; sucrose based (sugar cane), starch based (corn, wheat and barley) and lignocellulosic (which is mostly comprised of lignin, cellulose and hemicelluloses in grasses, wood and straw) materials. However, the limited supply of two biomass groups (sucrose and starch) will not satisfy society’s growing energy demands; thus biofuel technology based on lignocelluloses is under intense investigation. The main bottleneck in lignocellulosic biomass conversion for biofuel production is the enzymatic depolymerisation of cell wall polysaccharides into fermentable sugars. Protein engineering has recently been used to improve the performance of lignocelluloses degrading enzymes, as well as proteins involved in biofuel synthesis pathways. We have produced a recombinant enzyme that has the ability to produce monomeric sugars from a complex substrate. This presentation will summarize current efforts to develop an enzymatic treatment which would facilitate the economical processing of biomass available in Australia for bioenergy generation.

Production of green nanomaterials

While our awareness towards sustainable society and environment grows, the importance of ‘green’ materials and manufacturing is gaining significant recognition. We have demonstrated that naturally-occurring fibers as renewable raw materials can be converted into nanoparticles and nano fibers using simple top-down methods without introducing hazardous chemicals. This new class of green nanomaterials will have a wide range of environmental and biomedical applications owing to the inherent biocompatible, biodegradable and carbon-neutral nature.

Novel biorenewable plastics based on natural resources using ionic liquids

We report biorenewable plastics developed from natural resources such as cellulose, wool and microorganismsynthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer [1-3]. Novel materials were prepared by blending these natural polymers in an ionic liquid green solvent, 1-butyl-3-methylimidazolium chloride. Cellulose /PHBV blend materials were successfully prepared in this way. The ionic liquid was completely recycled with high yield and purity after the processing. The blend materials can be processed into different solid forms such as films, noodle-like fibers and bulk blocks. It was found that there exists hydrogen bonding interaction between the components which facilities the mixing of these polymers. The cellulose/PHBV blend materials all show phase-separated structure as revealed by micro ATR-FTIR imaging (Figure 1) and scanning electron microscopy (SEM). The PHBV domains of 6 - 8 µm are distributed in a cellulose matrix at high concentrations of cellulose while the blend materials with high PHBV concentrations exhibit multiphase morphologies, including beadlike PHBV microdomains in the range of 300-400 nm. The dispersion of PHBV in cellulose leads to significant improvement in hydrophobicity due to its beadlike structure. The blend materials represent a class of degradable plastics from natural bioresources using the ionic liquid green solvent.

Drug release rate and anti-microbial effect of controlled-diffusion biopolymer membranes

In this study, a series of fibrous membranes made from cellulose acetate (CA) and polyester urethane (PEU) by co-electrospining or blend-electrospining were evaluated for drug release kinetics, in vitro anti-microbial activity and in vivo would healing performance when used as wound dressings. To stop common clinical infections, an antibacterial agent, Polyhexamethylene Biguanide (PHMB) was incorporated into e-spun fibres. The presence of CA in the wound healing membrane was found to improve hydrophilicity and permeability to air and moisture. The in vivo tests indicated that the addition of PHMB and CA considerably improved the wound healing efficiency. CA fibres became slightly swollen upon contacting with the wound exudates. It can not only speed up the liquid evaporation but also create a moisture environment for wound recovery. The drug release dynamics of membranes was controlled by the structure of membranes and component rations within membranes. The lower ration of CA:PEU retained the sound mechanical properties of membranes, and also reduced the boost release effectively and slowed down diffusion of antibacterial agent during in vitro tests. The controlled-diffusion membranes exert long-term anti-infective effect.

Electrospinning of nanofibres with parallel line surface texture for improvement of nerve cell growth

Nanofibres having a parallel line surface texture were electrospun from cellulose acetate butyrate solutions using a solvent mixture of acetone and N,N'-dimethylacetamide. The formation mechanism of the unusual surface feature was explored and attributed to the formation of voids on the jet surface at the early stage of electrospinning and subsequent elongation and solidification of the voids into a line surface structure. The fast evaporation of a highly volatile solvent, acetone, from the polymer solution was found to play a key role in the formation of surface voids, while the high viscosity of the residual solution after the solvent evaporation ensured the line surface to be maintained after the solidification. Based on this principle, nanofibres having a similar surface texture were also electrospun successfully from other polymers, such as cellulose acetate, polyvinylidene fluoride, poly(methyl methacrylate), polystyrene and poly(vinylidene fluoride-co-hexafluoropropene), either from the same or from different solvent systems. Polarized Fourier transform infrared spectroscopy was used to measure the polymer molecular orientation within nanofibres. Schwann cells were grown on both aligned and randomly oriented nanofibre mats. The parallel line surface texture assisted in the growth of Schwann cells especially at the early stage of cell culture regardless of the fibre orientation. In contrast, the molecular orientation within nanofibres showed little impact on the cell growth.

Performance of reverse osmosis (RO) for water recovery from permeates of membrane bio-reactor (MBR)

In this study, permeate from a hollow fiber polyethylene (PE) membrane bio-reactor (MBR) system treating synthetic agricultural wastewater was fed into a cellulose acetate brackish water reverse osmosis (BWRO30 2540) membrane system; three different trans-membranes pressures (TMPs) of 1000, 2500, and 4000 kPa were selected to evaluate the system performance in terms of general operating parameters as well as the removal of chosen important potential fouling water quality parameters. The results showed that highest corrected permeate flux rate was at a TMP of 2500 kPa, whereas lowest recorded at a TMP of 4000 kPa. Similar situation prevailed in water recovery rate. But temperature corrected specific fluxes decreased as the applied TMPs increased. In all selected TMPs, more than 96% of salinity was removed. Permeate from MBR as feed to reverse osmosis required frequent chemical cleaning than the microfiltration/ultrafiltration (MF/UF) permeates and granular media filter (GMF) filtered in order to maintain the required rate of product water. One of the reasons for this frequent chemical cleaning is due to higher total organic carbon as well as total nitrogen (TN) in the MBR permeate. This result needs to be further evaluated through field trials.

A cartilage-inspired lubrication system

Articular cartilage is an example of a highly efficacious water-based, natural lubrication system that is optimized to provide low friction and wear protection at both low and high loads and sliding velocities. One of the secrets of cartilage's superior tribology comes from a unique, multimodal lubrication strategy consisting of both a fluid pressurization mediated lubrication mechanism and a boundary lubrication mechanism supported by surface bound macromolecules. Using a reconstituted network of highly interconnected cellulose fibers and simple modification through the immobilization of polyelectrolytes, we have recreated many of the mechanical and chemical properties of cartilage and the cartilage lubrication system to produce a purely synthetic material system that exhibits some of the same lubrication mechanisms, time dependent friction response, and high wear resistance as natural cartilage tissue. Friction and wear studies demonstrate how the properties of the cellulose fiber network can be used to control and optimize the lubrication and wear resistance of the material surfaces and highlight what key features of cartilage should be duplicated in order to produce a cartilage-mimetic lubrication system.

Freshwater influences on hydrology and seagrass dynamics of intermittent estuaries

Many temperate estuaries have intermittently open and closed mouths, a feature that is often related to intermittent freshwater input. These systems, often overlooked due to their small size, can have large hydrological variability over medium-term time scales.

This variability presents potential difficulties for estuarine species particularly where anthropogenic alterations to freshwater flows can cause large deviations from natural patterns of tidal influence and inundation of habitat.

Influences of natural and hydrological variability on seagrasses were examined in two central Victorian estuaries with anthropogenically-modified but naturally-intermittent freshwater flows and mouth openings. Comparisons were focused on differences between an estuary with artificially-augmented freshwater inflow and an adjacent system, in which the volume and timing of inflows were altered by a reservoir. Eight additional estuaries in the region were also used to provide a context for these two main sites.

Hydrological changes during the three-year field component were affected by the ending of a drought and then a major flood a year later as well as by ongoing anthropogenic flow reduction and augmentation. These influences on hydrology were associated with an initially high seagrass coverage that was substantially reduced and showed signs of recovery only in the system that was affected by lower inflows. Such influences and responses also changed seasonally but to a much lesser extent than the responses to stochastic climatic events.

Natural flows were intermittent and varied substantially between years. Flooding flows represented up to 89% of the long-term annual average flow. Water quality was broadly typical of the region, with the exception of low pH in some tributaries, especially those of Anglesea estuary. Anthropogenic changes to flow were most evident at times of low natural flows and resulted in longer and more frequent periods of zero inflow to Painkalac estuary and a continual base flow to Anglesea. This base flow, from ponds containing coal ash, neutralised waters flowing from upstream and increased conductivity, except at times of high natural flow.

A three-state conceptual model of the magnitude and variability of water levels, based largely on the degree of tidal influence was identified and quantitatively assessed for the two estuaries that were the main focus of the study. These states in turn had a large influence on the area and inundation of benthic habitat. Floods tended to open the mouths of estuaries, which then remained tidal given sufficient flow to overcome sedimentary processes at the mouths. Low and zero inflow was a precondition for closure of the mouths of the estuaries. When closed, differences in inflow resulted in different endpoints in salinity patterns. From an initial pattern similar to a classic ‘salt wedge’, Painkalac estuary, with reduced inflow, quickly destratified and gradually became more saline, at times hypersaline. Anglesea estuary, with augmented flow, tended to remain stratified for longer until becoming completely fresh, given a long enough period of closure.

Episodic changes in the water quality of the estuaries were associated with different components of the freshwater flow regimes. At high flows, fresh waters of low pH with a high metal load entered Anglesea estuary. Except during the largest flood, when the estuary was completely flushed, this water was neutralised at the halocline and resulting in precipitation of metals. High flows into Painkalac were associated with elevated concentrations of clay-sourced suspended solids. During a closed period, with zero flow, a release of sediment-bound nutrients triggered by anoxia was observed in Painkalac, followed by an algal bloom.

The large decline in seagrass extent that was observed in both estuaries was closely related to floods and the subsequent reductions in potential habitat associated with the tidal states that followed. Analysis of historical patterns of extent against rainfall records suggested that periods of drought and extended mouth closures were related to establishment and expansion of beds. This model was similar to that described for South African estuaries and contrasted with more-seasonal patterns reported for local marine embayments.

Rates of in situ decomposition of seagrass detritus showed a mix of seasonal and disturbance-driven patterns of change, depending on estuary. Variability of these rates on a scale of 100s of metres was typically not significant, but there were a few episodes that were highly significant. A negative correlation between decomposition rate and seagrass extent was also observed. A novel technique for assessing cellulose decomposition potential in sediment, adapted from soil science, proved to be a useful tool for estuarine research. Results from this component of the study highlighted both small-scale variability that was inconsistent through time, and also stable differences in decomposition potential between depths and estuaries that were consistent with differences in hydrological state and salinity.

Given the relative lack of knowledge about processes in intermittent estuaries, particularly those relating to changes in freshwater inflow, results from this study will be of value both locally and for similar systems elsewhere. Locally, it is likely that flow regimes to both Anglesea and Painkalac estuaries will be reduced, following closure of the mine power station at Anglesea and due to increased demand from the reservoir above Painkalac. There is potential to manage flows from each of these sources to minimise downstream effects. Regionally, and globally, there are many intermittent estuaries in areas with Mediterranean-type climates. It has been predicted that the climates of these regions will become drier but with an increase in intensity of storm events, both of which have ramifications for flow regimes to estuaries. It is hoped that results of this study will contribute to more informed management of intermittent estuaries in the context of these likely changes.

What happens during natural protein fibre dissolution in ionic liquids

Here, we monitor the dissolution of several natural protein fibres such as wool, human hair and silk, in various ionic liquids (ILs). The dissolution of protein-based materials using ILs is an emerging area exploring the production of new materials from waste products. Wool is a keratin fibre, which is extensively used in the textiles industry and as a result has considerable amounts of waste produced each year. Wool, along with human hair, has a unique morphology whereby the outer layer, the cuticle, is heavily cross linked with disulphide bonds, whereas silk does not have this outer layer. Here we show how ILs dissolve natural protein fibres and how the mechanism of dissolution is directly related to the structure and morphology of the wool fibre. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Recycling textiles : The use of ionic liquids in the separation of cotton polyester blends

Textiles are commonly made from intimate blends of polyester and cotton, which makes recycling very difficult. We report for the first time the use of ionic liquid in the separation of polyester cotton blends. By selective dissolution of the cotton component, the polyester component can be separated and recovered in high yield. This finding presents an environmentally benign approach to recycling textile waste. © 2014 The Royal Society of Chemistry.