Envolvimento de Phytophthora cinnamomi no declínio de Quercus suber e Q. rotundifolia: estudo da influência de factores bióticos e abióticos na progressão da doença. Possibilidades de controlo químico do declínio

Tese dout., Ciências Agrárias, Universidade do Algarve, 2007

Study of transcripts and reactive oxygen species involved in the defence responses of Quercus suber against Phytophthora cinnamomi

The present work has the merit of exploring an insight into the activation of defence genes of Quercus suber during response to infection by Phytophthora cinnamomi. Thus, cDNA-AFLP methodology was used to identify gene fragments differentially present in the mRNA profiles of host cells of micropropagated Q. suber plantlets roots infected with zoospores of P. cinnamomi at different post challenge time points. Six candidate genes were selected based on their interesting cDNA-AFLP expression patterns and homology to genes known to play a role in defence. These six genes encode a cinnamyl alcohol dehydrogenase 2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), thaumatin-like protein (QsTLP), chitinase (QsCHI) and a 1,3-beta glucanase (QsGLU). The current work has been successful in evaluation of the expression of these genes by qRT-PCR. Data analysis revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the early hours of inoculation, while transcript profiles of thaumatin-like protein showed decreasing. No expression was detected for 1,3-beta-glucanase (QsGLU). Furthermore, the choice of suitable reference genes in any new experimental system is absolutely crucial in qRT-PCR; for this reason in this study and for the first time a set of potential reference genes were analyzed and validated for qRT-PCR normalization in the patho-system Phytophthora-Q. suber. Four candidate reference genes polimerase II (QsRPII), eukaryotic translation initiation factor 5A(QsEIF-5A), b-tubulin (QsTUB) and a medium subunit family protein of Clathrin adaptor complexes (QsCACs) were evaluated to determine the most stable internal references in Q. suber. Analysis of stability of genes was carried out using Genex software. Results indicated all these four potential reference genes assumed stable expression. Data analysis revealed that QsRPII and QsCACs were the two most stable genes, while genes QsTUB and QsEIF-5A were the third and the fourth most stable gene, respectively. In this study, a plasmid-based quantitative PCR method was developed to measure P. cinnamomi colonization during infection process of Q. suber. Plasmid-based detection of P. cinnamomi showed a gradual accumulation of the pathogen DNA in cork oak root tips up to 24 h post infection. The higher increase in P. cinnamomi/plasmid DNA ratio occurred between 18 and 24 h. One of the primary objectives of this research was to study the effect of cinnamomins (elicitins secreted by P. cinnamomin) on inducing defence mechanism against the pathogen, as recent histological and ultra-structural studies showed that P. cinnamomi was restricted to the outer cortex root fragments pre-treated with capsicien and cryptogein, suggesting that elicitins can stimulate plant defence reactions against P. cinnamomi. To complement these studies and to have a clear view of the nature of the interaction, the role of cinnamomins in the production of the oxidative burst [ROS and ROS scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)] and in the defence responses was evaluated. Cork oak seedlings were pretreated with alpha-cinnamomin and then inoculated with P. cinnamomi mycelia. Results showed a significant higher production of reactive oxygen species (ROS) (H2O2 and O2•-) in elicitin and non-elicitin treated roots in interaction with P. cinnamomi in comparison to the corresponding control. The plant group inoculated with the pathogen after cinnamomin treatment showed an earlier increase in H2O2 production but this was lower as compared with that group inoculated with P. cinnamomi alone. Also, in elicitin pre-treated group generally, a lower level of O2•− production during infection was observed as compared with inoculated roots with P. cinnamomi alone without elicitin treatment. Furthermore, in this study, we evaluated activities of antioxidant enzymes upon challenge with P. cinnamomi, with and without pretreatment with alpha cinnamomin. Results indicated that the activities of defense enzymes POD, SOD and CAT increased after P. cinnamomi inoculation when compared with those in the control group. Also, in the group treated with alpha-cinnamomin followed by P. cinnamomi inoculation, a higher level of enzymatic activities was detected as compared with elicitin non-treated group, which suggest the protective effect of alpha-cinnamomin against the pathogen due to higher elevated levels of defense enzymes POD, SOD and CAT during the infection period. Furthermore, a sensitive qPCR method was applied to measure the pathogen biomass in elicited and non-elicited Q. suber roots challenged with P. cinnamomi to elucidate the effect of cinnamomins on the colonization of P. cinnamomi. Plasmid-based quantification of P. cinnamomi showed a significant decrease in accumulation of the pathogen DNA in cork oak roots after treatment with alpha and beta-cinnamomins which attest the role of cinnamomins in promoting defense responses in cork oak against P. cinnamomi invasion.

Evaluation of the protective effect of Phlomis purpurea against Phytophthora cinnamomi in Fagaceae and of root metabolites involved

Tese de doutoramento, Ciências Agrárias (Proteção de Plantas), Faculdade de Ciência e Tecnologia, Universidade do Algarve, 2014

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.

Ecotypic variation in the response of Arabidopsis thaliana to Phytophthora cinnamomi

A variety of reactions to inoculation with Phytophthora cinnamomi ranging from high susceptibility to moderate resistance were found in 20 ecotypes of Arabidopsis thaliana. P</i>. cinnamomi zoospores successfully colonised both root and leaf tissue of Arabidopsis and sporulation in the form of chlamydospores and sporangia occurred in leaves and roots of each ecotype but the number varied considerably between ecotypes. In the more susceptible ecotypes, colonisation was characterised by rapid intercellular growth and sporulation of the pathogen from 48 h post inoculation. In less susceptible ecotypes, P. cinnamomi was limited to a defined region within tissues. In response to P. cinnamomi infection, several ecotypes expressed active defence responses in both root and leaf tissue. Callose formation was closely associated with lesion restriction as was the production of the reactive oxygen species, hydrogen peroxide. The oxidative burst was not limited to the site of pathogen ingress but also occurred in distant, uninfected tissues. We have characterised an Arabidopsis–P. cinnamomi system that will be useful for further studies of active resistance mechanisms.

Spatial model for predicting the presence of cinnamon fungus (Phytophthora cinnamomi) in sclerophyll vegetation communities in south-eastern Australia

The pathogen Phytophthora cinnamomi causes extensive 'dieback' of Australian native vegetation. This study investigated the distribution of infection in an area of significant sclerophyll vegetation in Australia. It aimed to determine the relationship of infection to site variables and to develop a predictive model of infection. Site variables recorded at 50 study sites included aspect, slope, altitude, proximity to road and road characteristics, soil profile characteristics and vegetation attributes. Soil and plant tissues were assayed for the presence of the pathogen. A geographical information systyem (GIS) was employed to provide accurate estimations of spatial variables and develop a predictive model for the distribution of P. cinnamomi. The pathogen was isolated from 76% of the study sites. Of the 17 site variables initially investigated during the study a logistic regression model identified only two, elevation and sun-index, as significant in determining the probability of infection. The presence of P. cinnamomi infection was negatively associated with elevation and positively associated with sun-index. The model predicted that up to 74% of the study area (11 875 ha) had a high probability of being affected by P. cinnamomi. However, the present areas of infection were small, providing an opportunity for management to minimize spread into highly susceptible uninvaded areas.

Potassium phosphonate alters the defence response of Xanthorrhoea australis following infection by Phytophthora cinnamomi

Potassium phosphonate (phosphite) is widely used in the management of Phytophthora diseases in agriculture, horticulture and natural environments. The Austral grass tree, Xanthorrhoea australis, a keystone species in the dry sclerophyll forests of southern Australia, is susceptible to Phytophthora cinnamomi, but is protected by applications of phosphite. We examined the effect of phosphite application on the infection of X. australis seedlings and cell suspension cultures by zoospores of P. cinnamomi. Phosphite induced more intense cellular responses to pathogen challenge and suppressed pathogen ingress in both seedlings and cell cultures. In untreated X. australis seedlings, hyphal growth was initially intercellular, became intracellular 24 h after inoculation, and by 48 h had progressed into the vascular tissue. In phosphite-treated seedlings, growth of P. cinnamomi remained intercellular and was limited to the cortex, even at 72 h after inoculation. The cell membrane retracted from the cell wall and phenolic compounds and electron dense substances were deposited around the wall of infected and neighbouring cells. Suspension cells were infected within 6 h of inoculation. Within 24 h of inoculation, untreated cells were fully colonised, had collapsed cytoplasm and died. The protoplast of phosphite-treated suspension cells collapsed within 12 h of inoculation, and phenolic material accumulated in adjacent, uninfected cells. No anatomical response to phosphite treatment was observed before infection of plant tissues, suggesting that the phosphite-associated host defence response is induced following pathogen challenge. Anatomical changes provide evidence that phosphite stimulates the host defence system to respond more effectively to pathogen invasion.

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.

Elucidation of defence responses and signalling pathways induced in Arabidopsis thaliana following challenge with Phytophthora cinnamomi

Arabidopsis thaliana (Arabidopsis) Col-0 was inoculated with Phytophthora cinnamomi to assess the interaction and defence responses involved. Pathogen ingress and asexual reproduction occurred on root tissue but not leaf tissue. The colonisation of root tissue did not cause disease symptoms or plant death, indicating that Arabidopsis Col-0 was tolerant of the infection. The induction of several plant defence responses including the expression of defence-related genes were found, with differences displayed between inoculated root and leaf tissue. Arabidopsis defence-related gene mutant/over-expressing lines were also inoculated with P. cinnamomi but none of the lines tested exhibited a marked increase in susceptibility to the pathogen.

Phytophthora cinnamomi and Australia’s biodiversity : impacts, predictions and progress towards control

Phytophthora cinnamomi continues to cause devastating disease in Australian native vegetation and consequently the disease is listed by the Federal Government as a process that is threatening Australia’s biodiversity. Although several advances have been made in our understanding of how this soil-borne pathogen interacts with plants and of how we may tackle it in natural systems, our ability to control the disease is limited. The pathogen occurs widely across Australia but the severity of its impact is most evident within ecological communities of the south-west and south-east of the country. A regional impact summary for all states and territories shows the pathogen to be the cause of serious disease in numerous species, a significant number of which are rare and threatened. Many genera of endemic taxa have a high proportion of susceptible species including the iconic genera Banksia, Epacris and Xanthorrhoea. Long-term studies in Victoria have shown limited but probably unsustainable recovery of susceptible vegetation, given current management practices. Management of the disease in conservation reserves is reliant on hygiene, the use of chemicals and restriction of access, and has had only limited effectiveness and not provided complete control. The deleterious impacts of the disease on faunal habitat are reasonably well documented and demonstrate loss of individual animal species and changes in population structure and species abundance. Few plant species are known to be resistant to P. cinnamomi; however, investigations over several years have discovered the mechanisms by which some plants are able to survive infection, including the activation of defence-related genes and signalling pathways, the reinforcement of cell walls and accumulation of toxic metabolites. Manipulation of resistance and resistance-related mechanisms may provide avenues for protection against disease in otherwise susceptible species. Despite the advances made in Phytophthora research in Australia during the past 40 years, there is still much to be done to give land managers the resources to combat this disease. Recent State and Federal initiatives offer the prospect of a growing and broader awareness of the disease and its associated impacts. However, awareness must be translated into action as time is running out for the large number of susceptible, and potentially susceptible, species within vulnerable Australian ecological communities.

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 (&kappa; = 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.

Effect of Phytophthora cinnamomi on the habitat utilization of Antechinus stuartii in a Victorian forest

Phytophthora cinnamomi (Cinnamon fungus) is a pathogenic soil fungus which infects plant communities along the south-eastern coast of Australia, and the south-western corner of Western Australia. The symptoms of this disease include chlorosis, death of branches (ie. ‘dieback’), retarded growth and the eventual death of infected plants. This leads to devastating effects upon plant communities by altering both the structural and floristic characteristics of these communities. Small mammal species are dependent on specific features of their habitat such as vegetation structure and floristics. This thesis investigated alterations to the habitat of the insectivorous marsupial mouse, Antechinus stuartii, due to the presence of P. cinnamomi. The study was undertaken in an area of an open forest in the Brisbane Ranges, Victoria. Significant changes were found in both the floristic composition and structure of the vegetation at study sites infected with P, cinnamomi, compared to uninfected sites. The habitat utilization by A. stuartii of uninfected and infected vegetation was investigated using live trapping and radio-telemetric techniques. Capture rates were higher at sites uninfected by P. cinnamomi, and both male and females selected areas free from infection. Home range areas of males were significantly larger than those of females as assessed by telemetry. Both sexes spent a high proportion of time in areas dominated by Xanthorrhoea australis (Austral grass tree). There were significant relationships between the abundance of A. stuartii and the denseness of vegetation above 1 metre in height, and in particular, the proportion of cover afforded by X. australis. There were no significant differences in the cover of Eucalyptus spp. between uninfected and infected sites, but there were significantly more nest hollows in infected areas. The abundance of invertebrates was examined using pitfall traps. There were no significant differences in the abundance of the larger invertebrate taxa at infected and uninfected sites, but higher abundances of some micro-invertebrate groups in infected areas were recorded. The most likely factors considered to be influential in the habitat selection of A. stuartii were vegetation structure, and the presence of X. australis. To assess whether these factors were important the leaves of X. australis were removed with a brushcutter, to mimic the early effects of infection with P. cinnamomi. Animals did not respond to the alteration of vegetation structure in the short term (3-4 days). Longer-term experiments are required to assess the habitat utilization of A. stuartii at different periods following habitat manipulation. The implications of the presence of P. cinnamomi on the conservation of fauna are discussed. The destructive nature of the pathogen, and the slow rate of recovery from the disease, means that P. cinnamomi can be considered a threatening process to plant communities and the fauna that reside within that habitat. Future management of this disease within natural areas must therefore be cognisant of the potential of P. cinnamomi to significantly affect faunal as well as vegetative communities.

Use of phosphite as a control for Phytophthora cinnamomi in southeastern Victorian vegetation communities

One of the major aims of the research presented in this thesis was to assist managers of native vegetation communities in southeastern Australia in understanding the dynamics of P. cinnamomi with an important ecological species, Xanthorrhoea australis. It trialed the use of phosphite in large-scale field applications to establish the usefulness of this management option for the first time on Victorian flora. This thesis describes the process of disease development within mature X. Australia plants. For the first time it was shown that within X. australis plants, secondary disease symptoms are related to the percentage of stem that has been infested by the disease. It was evident that after initial invasion the pathogen moves via root xylem and throughout the plant within vascular to the stem, especially within the desmium. The research shows that the pathogen could not be isolated consistently even though it was considered to be responsible for disease symptoms. Trials of a control fungicide (Foli-R-fos 200) shows that protection occurs in many susceptible plants when 2 and 6g a.i./L phosphite is applied. Phytotoxicity occurred in native plants at Anglesea and within controlled environment trials when using ≥ 6g a.i./L. It will be shown that 2g a.i./L phosphite controls disease in sprayed plots within heathlands at Anglesea and a recently burnt coastal woodland community at Wilson’s Promontory. The proportion of healthy X. australis plants treated with phosphite was significantly higher than the proportion in control plots without phosphite. The research shows that phosphite was recovered from leaves of three species treated with Foli-R-fos 200 in the field. For the first time it has been shown that seed germination was reduced in two species when high concentrations of phosphite were applied. The first documentation of the effect that phosphite has on soil properties showed that nitrogen and oxidised organic carbon were the only parameters to alter significantly. This thesis provides answers to some important questions, answers that can now be used by managers in formulating better policies and actions at an operational level. There has been a dire need in Victoria to address many issues regarding P. cinnamomi and this thesis provides relevant and informative approaches to disease control, and a better understanding of the disease progress.

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.