Plant-pathogen interactions: toward development of next-generation disease-resistant plants

Briskly evolving phytopathogens are dire threats to our food supplies and threaten global food security. From the recent advances made toward high-throughput sequencing technologies, understanding of pathogenesis and effector biology, and plant innate immunity, translation of these means into new control tools is being introduced to develop durable disease resistance. Effectoromics as a powerful genetic tool for uncovering effector-target genes, both susceptibility genes and executor resistance genes in effector-assisted breeding, open up new avenues to improve resistance. TALENs (Transcription Activator-Like Effector Nucleases), engineered nucleases and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are breakthrough and powerful techniques for genome editing, providing efficient mechanisms for targeted crop protection strategies in disease resistance programs. In this review, major advances in plant disease management to confer durable disease resistance and novel strategies for boosting plant innate immunity are highlighted.

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.

Profiling secondary metabolites of plant roots inoculated with Phytophthora cinnamomi

The soil-borne pathogen, Phytophthora cinnamomi, continues to cause severe dieback in Australian native forest species and is of great international significance due to its global distribution. This research established a protocol to successfully identify phyto-chemicals associated with the defense response of plants challenged by the disease caused by Phytophthora cinnamomi.

Consensus guidelines for implementation of quality processes to prevent invasive fungal disease and enhanced surveillance measures during hospital building works, 2014

Healthcare-associated fungal outbreaks impose a substantial economic burden on the health system and typically result in high patient morbidity and mortality, particularly in the immunocompromised host. As the population at risk of invasive fungal infection continues to grow due to the increased burden of cancer and related factors, the need for hospitals to employ preventative measures has become increasingly important. These guidelines outline the standard quality processes hospitals need to accommodate into everyday practice and at times of healthcare-associated outbreak, including the role of antifungal stewardship programmes and best practice environmental sampling. Specific recommendations are also provided to help guide the planning and implementation of quality processes and enhanced surveillance before, during and after high-risk activities, such as hospital building works. Areas in which information is still lacking and further research is required are also highlighted.

Development of disease caused by Phytophthora cinnamomi in mature Xanthorrhoe australis

Over the past 30 years, heathland and open forest communities in south-eastern Australia dominated by Xanthorrhoea australis R.Br. have been severely affected by disease caused by Phytophthora cinnamomi Rands. The disease has caused a sharp decline in numbers of individuals within populations of X. australis; however, the etiology of the disease is unclear. The characteristics and disease symptoms induced by P. cinnamomi were analysed within nine mature X. australis plants that had been removed from the field. Seven plants showed typical disease symptoms that ranged from chlorotic leaves through to plant death. Plants showing disease symptoms had different numbers of infected roots, ranging from 0% in one dead plant, 40% infected roots in a plant showing yellowing of leaf tips and 67 and 86%, respectively, in two plants with severe chlorosis. There was variation within the roots, with some infected close to the stem while others were infected at more distal regions. Within stems of all plants, P. cinnamomi was difficult to isolate but was found in the desmium and stem apex and was associated with massive lesions within the central area of the stem. The symptoms of disease in X. australis are caused by a combination of damage to tissues of the roots and stem that may lead to a reduction in water and mineral transport throughout the plant.

Distribution of disease caused by Phytophthora cinnamomi in Wilsons Promontory National Park and potential for further impact

The extent of disease caused by Phytophthora cinnamomi was determined within vegetation communities of Wilsons Promontory National Park. Aerial survey of visible symptoms by helicopter and systematic survey along all roads and tracks followed by isolation of the pathogen from soil found that in total 551 ha of moist foothill forest, heath and heathy woodland broad vegetation types were affected by the disease. P. cinnamomi was isolated from 93% of sites that, based on the presence of visible symptoms, were expected to yield the pathogen. The species-rich heathy woodland was most affected with 6.5% of the total area of this type showing symptoms of disease. The size of infestation ranged from 229 ha on the slopes of the Vereker Range in the north to less than 1 ha along the Sealers Cove Walking Track in the south. The potential for disease to spread into uninfested vegetation was estimated for all sites from which P. cinnamomi was isolated. Eight of 18 sites where evidence of disease was found were estimated to have a high potential for further disease spread. This study indicates that even though the disease may be waning in some areas of the Park, the pathogen is active and easily isolated from others and provides a continuing threat to susceptible vegetation communities.

Public views of the benefits and barriers to the consumption of a plant-based diet

Objective: The aim of this study was to examine consumers' perceived benefits and barriers to the consumption of a plant-based diet. Design: Mail survey that included questions on perceived benefits and barriers to the consumption of a plant-based diet. Setting: Victoria, Australia. Subjects: Four hundred and fifteen randomly selected Victorian adults. Results: The main perceived barrier to adoption of a plant-based diet was a lack of information about plant-based diets (42% agreement). Sex, age and education differences were present in over a quarter of the barrier items. For example, non-university-educated respondents and older people were less willing to change their current eating pattern than were university educated and younger respondents. The main benefits associated with plant-based diets were health benefits, particularly decreased saturated fat intake (79% agreement), increased fibre intake (76%), and disease prevention (70%). Age, sex and education differences with regard to benefits were apparent, although sex differences were more important than age or education differences. Conclusions: The majority of respondents perceived there to be health benefits associated with the consumption of a plant-based diet. Compared with the proportion of respondents who agreed that there were particular benefits of eating a plant-based diet, perceived barriers were relatively low. An understanding of the perceived benefits and barriers of consuming a plant-based diet will help formulate strategies that aim to influence beliefs about plant foods, plant food consumption, and, ultimately, public health.

Potential microbial and chemical hazards to waterbirds at the Western Treatment Plant

The Western Treatment Plant, a major sewage treatment plant west of Melbourne, Australia, is widely regarded as a significant conservation site for waterbirds. But experiences from various parts of the world suggest that sewage can also be hazardous to waterbirds, and has probably been responsible for mass-kill events. The intent of this contribution is to raise awareness about the potential for adverse impacts of sewage treatment plants on waterbirds, and to stimulate debate on the issue, with the ultimate objective of developing appropriate management strategies to mitigate the risk of mass kills.

Investigation of plasmodiophora brassicae (clubroot disease) in vegetable brassica using arabidopsis thaliana as a model system

Clubroot, caused by Plasmodiophora brassicae, is the most devastating soil-borne disease of vegetable brassicas. It occurs all over the world and is responsible for crop losses of up to 10% every year. In Australia, the disease is being managed effectively with chemicals and cultural practices, but ideally control can be improved in the long term by the introduction of resistant cultivars. The life cycle ofP. brassicae and mode of action of plant resistance has not been fully elucidated because of the technical difficulties of working with an obligate, soil-borne plant pathogen. However, Arabidopsis thaliana, which is a host ofP. brassicae, has great potential as a model system for studying the life cycle, the infection process and development of resistance. We have developed a sand-liquid-culture system for growing Arabidopsis that allows easy observation of all life stages and, most importantly, the primary plasmodial stages within the root hair. The method was first optimised for observations of the lifecycle of the pathogen in a susceptible Arabidopsis ecotype (Col-3) where all stages of the lifecycle have now been observed and characterised. Further screening of Arabidopsis ecotypes for disease resistance has utilised one of the most virulent Australian pathotypes of brassica (ECD number 16/19/31). To date, Arabidopsis ecotype Ta-0 has shown a level of tolerance to the disease even though the roots get infected. It has been reported earlier that resistance toP. brassicae in Arabidopsis is due to one or a small number of genes. To examine changes in gene expression during the early, critical stages of infection, RNA was extracted from the susceptible and resistant ecotypes at two time points, 4 days and 17 days after inoculation. Microarray analysis will be used to investigate genome wide changes in gene expression during infection but also to identify candidate genes that may confer resistance to Australian isolates of the pathogen.

Defining plant resistance against Phytophthora Cinnamomi and application of resistance to revegetation

Phytophthora cinnamomi is a soil borne plant pathogen that causes devastating disease in many Australian ecosystems and threatens the survival of native flora. Compared with the number of plant species that are susceptible to P. cinnamomi, only a few species are known to be resistant and control of this pathogen by chemicals is difficult and undesirable in natural systems. The major aim of our research is therefore to characterise natural resistance and determine which signalling pathways and defence responses are involved. Our examination of resistance is being approached at several levels, one of which is through the use of the model plant, Arabidopsis. Previously, Arabidopsis had been shown to display ecotypic variation in responses to P. cinnamomi and we are exploring this further in conjunction with the analysis of a bank of Arabidopsis defence pathway mutants for their responses to the pathogen. These experiments will provide a fundamental basis for further analysis of the defence responses of native plants. Native species (susceptible and resistant) are being assessed for their responses to P. cinnamomi at morphological, biochemical and molecular levels. This research also involves field-based studies of plants under challenge at various sites throughout Victoria, Australia. The focus of this field-based research is to assess the responses of individual species to P. cinnamomi in the natural environment with the goal of identifying individuals within susceptible species that display 'resistance'. Understanding how plants are able to resist this pathogen will enable strategies to be developed to enhance species survival and to restore structure and biodiversity to the ecosystems under threat.

Plant pathogens causing vegetation dieback: a serious threat to the conservation of small mammals in Australia

The soil-borne plant pathogen Phytophthora cinnamomi occurs in most Australian states. It is pathogenic to many Australian species, particularly the Proteaceae, Fabaceae, Dillineaceae and Epacridaceae. In Western Australia, c. 2000 of the 9000 endemic plant species are directly affected by the disease. The epidemic of plant deaths caused by P. cinnamomi is recognised as one of 11 Key Threatening Processes to the Australian Environment, and is now also acknowledged as a potential threat fauna in a range of communities. The implications of landscape modification due to the effects of P. cinnamomi dieback prompted our research, designed to measure the distribution and abundance of small mammals in disease-affected ecosystems. This study was in the Jarrah (Eucalyptus marginata) forests in the Darling Range, Western Australia and measured the distribution and abundance of one small mammal species, the Mardo (Antechinus flavipes) by Elliott trapping in forests with (1) high, (2) mixed and (3) no evidence of Phytophthora dieback. Trap success was highest in sites with no effect of Phytophthora (7.3 animals per 100 trap nights), whereas the lowest trap success was recorded at the high impact sites (0.67 animals per 100 trap night). There was a significant difference in trap success of Mardos in Elliott trapping over 1800 trap nights (x²= 23.19, d.f = 5, p < 0.001). An examination of the distribution of individuals and sexes suggests that Phytophthora-affected sites act as sinks for Mardos, while source areas are healthy, unaffected Jarrah forest.

Development and use of a model system to monitor clubroot disease progression with an Australian field collection 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-0 known to 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.

Use of high resolution digital multi-spectral imagery to assess the distribution of disease caused by Phytophthora cinnamomi on heathland at Anglesea, Victoria

Disease caused by the soilborne plant pathogen Phytophthora cinnamomi causes long-term floristic and structural changes in native vegetation communities in Australia. Key components of the management of this disease are to know where it occurs and the rate at which it spreads. The distribution of P. cinnamomi has generally been assessed as locality points of infestation and mapping the extent of diseased vegetation in any area is difficult and costly. This study was undertaken in P. cinnamomi-infested heathland communities in southern Victoria, Australia, where the symptoms of P. cinnamomi arise as a mosaic within healthy vegetation. We investigated the potential to improve the efficiency and effectiveness of mapping and monitoring vegetation affected by P. cinnamomi using digital multi-spectral imaging. This technique was developed for the purposes of monitoring vegetation and provides a single, seamless ortho-rectified digital image over the total area of interest. It is used to spatially quantify small differences in the characteristics of vegetation. In this study, the symptoms of disease caused by P. cinnamomi infestation were related to differences in the imagery and were used to map areas of infestation. Comparison of the digital multi-spectral imaging indications with on-ground observations gave moderate accuracy between the datasets (κ = 0.49) for disease and healthy indications. This study demonstrates the ability of the technique to determine disease extent over broad areas in native vegetation and provides a non-invasive, cost effective tool for management.

Abscisic acid regulation of plant defence responses during pathogen attack

The plant hormone, abscisic acid (ABA), has previously been shown to have an impact on the resistance or susceptibility of plants to pathogens. In this thesis, it was shown that ABA had a regulatory effect on an extensive array of plant defence responses in three different plant and pathogen interaction combinations as well as following the application of an abiotic elicitor. In unique studies using ABA deficient mutants of Arabidopsis, exogenous ABA addition or ABA biosynthesis inhibitor application and simulated drought stress, ABA was shown to have a profound effect on the outcome of interactions between plants and pathogens of differing lifestyles and from different kingdoms. The systems used included a model plant and an important agricultural species: Arabidopsis thaliana (Arabidopsis) and Peronospora parasitica (a biotrophic Oomycete pathogen), Arabidopsis and Pseudomonas syringae pathovar tomato (a biotrophic bacterial pathogen) and an unrelated plant species, soybean (Glycine max) and Phytophthora sojae (a hemibiotrophic Oomycete pathogen), Generally, a higher than basal endogenous ABA concentration within plant tissues at the time of avirulent pathogen inoculation, caused an interaction shift towards what phenotypically resembled susceptibility. Conversely, a lower than basal endogenous ABA concentration in plants inoculated with a virulent pathogen caused a shift towards resistance. An extensive suppressive effect of ABA on defence responses was revealed by a range of techniques that included histochemical, biochemical and molecular approaches. A universal effect of ABA on suppression or induction of the phenylpropanoid pathway via regulation of the key entry point gene, phenylalanine ammonia-lyase (PAL), when stimulated by biotic or abiotic elicitors was shown. ABA also influenced a wide variety of other defence-related components such as: the development of a hypersensitive response (HR), the accumulation of the reactive oxyden species, hydrogen peroxide and the cell wall strengthening compounds lignin and callose, accumulation of SA and the phytoalexin, glyceollin and the transcription of the SA-dependent pathogenesis- related gene (PR-1). The near genome-wide microarray gene expression analysis of an ABA induced susceptible interaction also revealed an yet unprecedented insight into the great diversity of defence responses that were influenced by ABA that included: disease resistance like proteins, antimicrobial proteins as well as phenylpropanoid and tryptophan pathway enzymes. Subtle differences were found in the number and type of defence responses that were regulated by ABA in each type of plant and pathogen interaction that was studied. This thesis has clearly identified in plant/pathogen interactions previously unknown and important roles for ABA in the regulation of many defence responses.