Robert Faggian

Specific polymerase chain reaction primers for the detection of Plasmodiophora brassicae in soil and water.

ABSTRACT The development of specific oligonucleotide primers for Plasmodiophora brassicae has led to a nested polymerase chain reaction (PCR) detection method for P. brassicae in soil and water. Initially, the PCR was used to amplify a section of the rDNA repeat. The PCR products were sequenced and the data used to design primers that were directed at the ribosomal RNA genes and internal transcribed spacer regions. Specificity was tested against more than 40 common soil organisms, host plants, and spore suspension contaminants, as well as P. brassicae isolates from around Australia and the world. Sensitivity was determined to be 0.1 fentograms (fg; 10(-15) g) for pure template and as low as 1,000 spores per g of potting mix. In soil, P. brassicae was detected in all soils where the inoculum was sufficient to result in clubroot symptoms. Also outlined is a simple method of DNA extraction from soil.

Persistence of Escherichia coli on injured iceberg lettuce in the field, overhead irrigated with contaminated water.

Fresh produce is increasingly implicated in food-related illnesses. Escherichia coli can survive in soil and water and can be transferred onto plant surfaces through farm management practices such as irrigation. A trial was conducted to evaluate the impact of field conditions on E. coli persistence on iceberg lettuce irrigated with contaminated water, and the impact of plant injury on the persistence of E. coli. Lettuce heads were injured at 14, 7, 3, 2, 1, and 0 days before inoculation, with uninjured heads used as a control. All lettuce heads (including controls) were overhead irrigated with a mixture of nonpathogenic E. coli strains (10(7) CFU/ml). E. coli counts were measured on the day of inoculation and 5 days after, and E. coli was detected on all lettuce head samples. Injury immediately prior to inoculation and harvest significantly (P = 0.00067) increased persistence of E. coli on lettuce plants. Harsh environmental conditions (warm temperatures, limited rainfall) over 5 days resulted in a 2.2-log reduction in E. coli counts on uninjured lettuce plants, and lettuce plants injured more than 2 days prior to inoculation had similar results. Plants with more recent injuries (up to 2 days prior to inoculation) had significantly (P = 7.6 x 10(-6)) greater E. coli persistence. Therefore, growers should postpone contaminated water irrigation of lettuce crops with suspected injuries for a minimum of 2 days, or if unavoidable, use the highest microbiological quality of water available, to minimize food safety risks.

Analysis of global host gene expression during the primary phase of the Arabidopsis thaliana–Plasmodiophora brassicae interaction

Microarray analysis was used to investigate changes in host gene expression during the primary stages of the interaction between the susceptible plant Arabidopsis thaliana (L.) Heynh ecotype Col-0 and the biotrophic pathogen Plasmodiophora brassicae Woronin. Analyses were conducted at 4, 7 and 10 days after inoculation (DAI) and revealed significant induction or suppression of a relatively low number of genes in a range of functional categories. At 4 DAI, there was induced expression of several genes known to be critical for pathogen recognition and signal transduction in other resistant host-pathogen interactions. As the pathogen further colonised root tissue and progressed through the primary plasmodium stage to production of zoosporangia at 7 and 10 DAI, respectively, fewer genes showed changes in expression. The microarray results were validated by examining a subset of induced genes at 4 DAI by quantitative real-time reverse transcriptase PCR (RT-qPCR) analysis all of which correlated positively with the microarray data. The two A. thaliana mutants jar1 and coiI tested were found to be susceptible to P. brassicae. The involvement of defence-related hormones in the interaction was further investigated and the findings indicate that addition of salicylic acid can suppress clubroot disease in A. thaliana plants.

Detection and Measurement of Plasmodiophora brassicae

Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of brassicas. Management of clubroot is difficult, and the best means of avoiding the disease include planting in areas where P. brassicae is not present and using plants and growing media free from pathogen inoculum. As P. brassicae is not culturable, its detection has traditionally relied on plant bioassays, which are time-consuming and require large amounts of glasshouse space. More recently, fluorescence microscopy, serology, and DNA-based methods have all been used to test soil, water, or plant samples for clubroot. The use of fluorescence microscopy to detect and count pathogen spores in the soil requires significant operator skill and is unlikely to serve as the basis for a routine diagnostic test. By contrast, serologic assays are inexpensive and amenable to high-throughput screening but need to be based on monoclonal antibodies because polyclonal antisera cannot be reproduced and are therefore of limited quantity. Several polymerase chain reaction (PCR)-based assays have also been developed; these are highly specific for P. brassicae and have been well-correlated with disease severity. As such, PCR-based diagnostic tests have been adopted to varying extents in Canada and Australia, but wide implementation has been restricted by sample processing costs. Efforts are underway to develop inexpensive serologic on-farm diagnostic kits and to improve quantification of pathogen inoculum levels through real-time PCR. Proper detection and quantification of P. brassicae will likely play an increasingly important role in the development of effective clubroot management strategies.

Creating communities of practice: scoping purposeful design

Purpose – This paper seeks to scope the nature and form of practices, understandings and institutional arrangements thatmight contributetothe successful ‘‘design’’ andcontinuity of Communities of Practice (CoP) in a state government department in Australia. The study aims to provide research evidence to support the design and establishment of a CoP based on systems thinking within this department. Design/methodology/approach – A total of 13 semi-structured interviews were undertaken involving 14 informants. The interviewer also attended one CoP meeting. An emergent approach to research design was adopted with data analysis guided by previous studies on CoPs. Findings – The research revealed the existence of six CoPs that were purposefully created internally by the department. Six ‘‘design’’ and practice considerations were suggested for practitioners aiming to create and sustain successful CoPs. Research limitations/implications – Interview material was the only source of primary data and it was gathered from one organisation only – a state government department in Australia. Findings indicate that the role of the CoP coordinator is still not fully understood. Practical implications – The results from this study can be used in re-designing a systems thinking CoP to support systems thinking within the department. The study also revealed that purposefully designing CoPs is possible and useful for practitioners aiming to collaborate and share expertise across disciplinary and divisional boundaries. Originality/value – This study provides some guidance for the purposeful design of CoPs, which has been under-examined in the literature.

Occurrence of antibiotic resistance genes in reclaimed water and river water in the Werribee Basin, Australia

The purpose of this study was to investigate the occurrence of antibiotic resistance genes (ARGs) in water used for irrigation in the Werribee River Basin, Australia, including river water and reclaimed effluent water (reclaimed water). Samples of reclaimed water, collected over a one-year period, were screened for the occurrence of ARGs using PCR detection assays. The presence of ARGs in the reclaimed water samples were contrasted with that of water samples taken from the Werribee River Basin, collected over the same time period, from five points selected for varying levels of urban and agricultural impact. Of the 54 river water samples collected, 2 (4%), 2 (4%), 0 and 0 were positive for methicillin, sulfonamide, gentamicin and vancomycin-resistant genes, respectively, while 6 of 11 reclaimed water samples were positive for methicillin (9%) and sulfonamide (45%). The presence/absence of ARGs did not appear to correlate with other measured water quality parameters. The low detection of ARGs in river water indicates that, regardless of its poor quality, the river has not yet been severely contaminated with ARGs. The greater prevalence of ARGs in reclaimed water indicates that this important agricultural water source will need to be monitored into the future.

Development and use of a model system to monitor clubroot disease progression with an Australian field population of Plasmodiophora brassicae.

A modified sand-liquid culture method facilitated easy visualisation of the primary life cycle stages of Plasmodiophora brassicae within clean root hairs of the Arabidopsis host. Pathogen penetration occurred from day 4 onwards and then primary plasmodia developed within the host root. Several Arabidopsis ecotypes tested in varying growth conditions showed differences in disease expression. Defined growth cabinet conditions were found most suitable for studying disease progression in the ecotypes and for achieving uniform infection and disease development. Arabidopsis ecotypes Ta-0 and Tsu-0knownto be partially resistant to a German single-spore isolate of P. brassicae were susceptible to an Australian (Victorian) field population of P. brassicae. The European clubroot differential test was used to confirm virulence and describe the pathotype of the Victorian field population. Knowledge of the interaction of an Australian population of P. brassicae with its host will provide valuable information on a disease which is very difficult to control.

Evaluation of Deterministic and Complex Analytical Hierarchy Process Methods for Agricultural Land Suitability Analysis in a Changing Climate

Land suitability analysis is employed to evaluate the appropriateness of land for a particular purpose whilst integrating both qualitative and quantitative inputs, which can be continuous in nature. However, in agricultural modelling there is often a disregard of this contiguous aspect. Therefore, some parametric procedures for suitability analysis compartmentalise units into defined membership classes. This imposition of crisp boundaries neglects the continuous formations found throughout nature and overlooks differences and inherent uncertainties found in the modelling. This research will compare two approaches to suitability analysis over three differing methods. The primary approach will use an Analytical Hierarchy Process (AHP), while the other approach will use a Fuzzy AHP over two methods; Fitted Fuzzy AHP and Nested Fuzzy AHP. Secondary to this, each method will be assessed into how it behaves in a climate change scenario to understand and highlight the role of uncertainties in model conceptualisation and structure. Outputs and comparisons between each method, in relation to area, proportion of membership classes and spatial representation, showed that fuzzy modelling techniques detailed a more robust and continuous output. In particular the Nested Fuzzy AHP was concluded to be more pertinent, as it incorporated complex modelling techniques, as well as the initial AHP framework. Through this comparison and assessment of model behaviour, an evaluation of each methods predictive capacity and relevance for decision-making purposes in agricultural applications is gained.

Screening for resistance to potato cyst nematode in Australian potato cultivars and alternative solanaceous hosts

The potato cyst nematodes (PCN), Globodera rostochiensis (Woll.) and G. pallida (Stone), are major pests of ware and seed potato (Solanum tuberosum L.) crops worldwide and severely impact the movement of potatoes around the globe through quarantine restrictions. In Australia, only G. rostochiensis has been discovered, on four separate occasions between 1986 and 2008. The infested areas are the subject of strict regulation and quarantine procedures and while they are considered to be contained, managing nematode populations remains a priority. This study has identified the G. rostochiensis Ro1 resistance-status of potato cultivars currently grown by Australian potato growers, and new cultivars emerging from the Australian Potato Breeding Program. Resistance was assessed by a simple and robust procedure carried out in a purpose-built quarantine facility. Of the 24 potato cultivars grown in the affected Koo Wee Rup district in 2004, 10 were resistant to nematode infestation, including the locally important cultivar Atlantic. Other cultivars important to the Victorian and Australian potato industry, such as Kennebec, Desiree, Sebago and Coliban, were classified as susceptible. Importantly, this study provided evidence that the Koo Wee Rup PCN population was able to complete its lifecycle on the native plant species, S. aviculare (kangaroo apple), potentially acting as an alternate host and spreading PCN among potato crops.

Designing resilient regions by applying Blue-Green Infrastructure concepts

In Australia, weather extremes (droughts and floods) are an accepted component of coupled human-environment systems. Australia is the driest inhabited continent on earth and also has the greatest annual rainfall and run-off variability. Competition for water between the environment, agriculture and domestic uses is intense and the cause of much public debate. It is not unusual for parts of Australia to transition quickly from a state of extreme water scarcity to one of severe flooding. In fact, floods cause more damage in Australia than any other natural disaster. Climate change will exacerbate the situation through increased frequency and intensity of heavy rainfall events and also more intense and longer-lasting droughts. The combination of drought followed by intense rainfall increases the risk of severe flooding, with impacts on civil infrastructure (road and bridge washouts, damage to houses), and impacts on agriculture (soil erosion and destruction of crops and livestock). Structural flood mitigation activities in Australia, such as the construction of levees, was initially driven by private landholders. These measures were often not well planned or integrated at larger scales and therefore have been viewed with some suspicion. More recently, non-structural (land planning, emergency management) approaches have become the key flood mitigation measure. In contrast, The Netherlands takes a structural approach through concepts like Blue-Green Infrastructure (BGI), with the aim of “giving the flood a pathway”. In this context, structural interventions in the landscape provide alternative pathways for flood water, slowing the waters progress such that flood damage is mitigated. Our research focuses on the feasibility of implementing BGI in Australia, considering the costs and benefits in terms of the biophysical environment, infrastructure and socio-economic systems, in order to increase the resilience of rural and regional communities. The research will inform strategic and statutory planning at the regional level.