Thesis (PhD)--Stellenbosch University, 1983. / ENGLISH ABSTRACT: No abstract available / AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar

Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains viii, 56 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 52-56).

Phytophthora cinnamomi Rands is a common and devastating pathogen of cultivated proteas worldwide. Webb (1997) described a Sudden Death plant disease of proteas in Western Australia (WA) protea plantations. Proteas that suffer the syndrome display symptoms such as stunted growth, wilting, chlorosis and often death. In the current study, a number of protea plantations in the southwest of WA were visited to quantify the extent that P. cinnamomi was attributing to deaths of cultivated proteas. The survey indicated that P. cinnamomi is the major cause of Sudden Death in proteas. A range of other fungi (Fusarium, Botryosphaeria, Pestalotiopsis, Alternaria) and pests (nematodes, mealy bug, scale insects) were also identified to be contributing to protea death and decline in WA plantations. In many cases the factors contributing to protea disease appeared complex, with a range of physical factors or nutritional imbalances commonly associated with these pathogens and pests. As P. cinnamomi was the major cause of death of cultivated proteas the remainder of the experiments described in this dissertation investigated its control in horticultural plantings. Biofumigation has the potential to become an important technique in an overall integrated management approach to P. cinnamomi. In this thesis, biofumigation refers to the suppression of pathogens and pests by the incorporation of Brassica plants into the soil. Two biofumigants (Brassica juncea (L.) Czern., B. napus L.) were screened for their effect on the in vitro growth of five common Phytophthora species (P. cinnamomi, P. cactorum (Lebert & Colin) Schroeter., P. citricola Sawada, P. cryptogea Pethyb. & Laff. and P. megasperma Drechsler). Growth was determined by the measuring dry weight and radial growth of vegetative hyphae. B. juncea was found to be superior in its suppressive effect compared to B. napus. There was also significant variation in the sensitivity of the Phytophthora species to the suppressive effects of the biofumigants. P. cinnamomi was the most sensitive of the five species investigated. Where the rates of the biofumigant were sufficient to suppress growth of Phytophthora, the suppressive effect was mostly fungicidal. To determine how B. juncea and B. napus affect the infective ability and survival of P. cinnamomi, their effects on sporangia and chlamydospores production in soil was investigated in vitro. P. cinnamomi colonised Miracloth discs were added to soil amended with the two Brassica species, before being removed every two days over an eight day period for the determination of sporangia production, chlamydospore production and infective ability. Only the soils amended with B. juncea significantly reduced sporangia production in P. cinnamomi. Both Brassica species increased the percentage of aborted or immature sporangia and reduced the infective ability of the pathogen. Neither Brassica species had any effect on zoospore release or chlamydospore production in P. cinnamomi. Soil cores and soil leachate were collected from biofumigant-amended field soils to determine the inoculum potential and infective ability of the pathogen under glasshouse conditions. Amending the soil with both Brassica species had an immediate suppressive effect on the inoculum potential and infective ability of the P. cinnamomi. However, after this initial suppression there was a gradual increase in the recovery of the pathogen over the monitoring period of four weeks. To determine if the suppression would result in decreased disease incidence in a susceptible host, Lupinus angustifolius L. seeds were planted in the biofumigant amended soil. B. juncea amended soils reduced the disease incidence of P. cinnamomi by 25%. B. napus had no effect on disease incidence in L. angustifolius. Although the current study had demonstrated that biofumigants could suppress the growth, sporulation and infection of P. cinnamomi, it was unclear if this would equate to a reduction in disease incidence when applied in the field. A field trial was conducted on a protea plantation in the southwest of Western Australia that compared biofumigation with B. juncea to chemical fumigation (metham sodium) and soil solarisation. The three soil treatments were used in an integrated management approach to control P. cinnamomi that included the use of a hardwood compost, mulch and water sterilisation. All treatments were monitored during their application to ensure the treatments were conducted successfully. The three soil treatments significantly reduced the recovery of the pathogen and the infective ability of the pathogen to a soil depth of 20 cm. Metham sodium was the most suppressive soil treatment and soil solarisation was the least suppressive treatment. Only the metham sodium treatment resulted in a significant reduction in the incidence of root rot in Leucadendron salignum P.J. Bergius x laureolum (Lam.) Fourc (c.v. Safari Sunset) over the monitoring period of three years. Another field trial was conducted on the same protea plantation to compare the effectiveness of B. juncea and B. napus, without the use of other control strategies, to reduce the incidence of P. cinnamomi infection of Leucadendron Safari Sunset. The concentration of isothiocyanates was monitored for seven days after the incorporation of the biofumigants. Although both Brassica species reduced the recovery and infective ability of the pathogen, neither biofumigant reduced the incidence of root rot in Leucadendron Safari Sunset. In conclusion, P. cinnamomi is the most common and devastating pathogen in WA protea plantations. The current study demonstrated that P. cinnamomi is sensitive to the suppressive nature of biofumigants. Biofumigants can suppress the in vitro growth, sporulation, infective ability of P. cinnamomi and reduce the incidence of the disease caused by the pathogen in the glasshouse. Of the two Brassica species investigated, B. juncea was superior in its ability to control P. cinnamomi compared to B. napus. When applied in the field, biofumigation using B. juncea was found to be more suppressive that soil solarisation, but not as effective as metham sodium.

Phytophthora cinnamomi is an introduced soilborne phytopathogen to Western Australia (WA) and impacts on 2000 of the approximately 9000 plant species indigenous in the southwest of WA. Amongst these is Eucalyptus marginata (jarrah), the dominant and economically important hardwood timber species of the jarrah forest. This thesis aimed to investigate the morphological, pathogenic and genotypic variation in two local WA populations of P. cinnamomi isolates. The populations were selected from areas where jarrah clonal lines selected for resistance to P. cinnamomi may be used in the rehabilitation of infested jarrah forest and rehabilitated bauxite minesites in the southwest of WA. Resistance against a range of isolates using different inoculation methods. Seventy-three isolates of P. cinnamomi were collected from diseased jarrah and Corymbia calophylla (marri) trees from two populations located 70 km apart and these were examined for phenotypic and genotypic variation. Microsatellite DNA analysis showed that all isolates were of the same clonal lineage. In P. cinnamomi for the first time I show that there is a broad and continuous variation in the morphology and pathology between two populations of one clonal lineage, and that all phenotypes varied independently from one another. No relationship was found between morphological and pathogenic characters. The ability of isolates in both populations to cause deaths ranged from killing all plants within 59 days to plants being symptomless 182 days after inoculation. Single and multiple paragynous antheridia formed along with amphigynous ones in mating studies with all WA isolates and a sample of worldwide isolates. Developmental studies and cytological examination showed fertilisation tubes developed asynchronously or synchronously from both antheridial types and indicated that either antheridial type contributed a nucleus for fertilisation of the oosphere. This is the first report of paragynous antheridial associations in P. cinnamomi. Antheridial variation is a characteristic that needs to be adjusted in the taxonomic Phytophthora identification keys. In underbark and zoospore stem inoculations of three 1.5-year-old jarrah clonal lines (two ranked as resistant (RR) and one as susceptible (SS) to P. cinnamomi in the original selection trials) at 15, 20, 25 and 30°C, it was found that the method of inoculation did not produce comparable results, particularly at 25 and 30°C. At these temperatures, all three clonal lines had 100% mortality when inoculated underbark, but when inoculated with zoospores, one RR line had 60% survival and the SS and remaining RR line had 100% mortality. Generally, the level of resistance of all clonal lines declined with increasing temperature. Lesion development was measured at 20, 25 and 30°C for 4 days in detached branches of an RR and SS clonal line inoculated underbark with four different P. cinnamomi isolates. Detached branches were found to be a potential screen for jarrah resistance to P. cinnamomi and to allow the identification of susceptible and resistant clonal lines at 30°C. Lesion and colonisation development of P. cinnamomi isolates were assessed in situ (late autumn) of seed-grown and clonal lines of 3.5 to 4.5 year-old jarrah trees growing in a rehabilitated minesite jarrah forest in underbark inoculation of lateral branches (1995) or simultaneously in lateral branches and lateral roots (1996). Trees were underbark inoculated in lateral branches and lateral roots. Colonisation was more consistent as a measure of resistance than lesion length over the two trials because it accounted for the recovery of P. cinnamomi from macroscopically symptomless tissue beyond lesions, which on some occasions, was up to 6 cm. In the two trials, one RR clonal line consistently had small lesion and colonisation lengths in branches and roots. In contrast, the remaining two RR clonal lines had similar lesion and colonisation lengths to the SS clonal line and may, therefore, not be suitable for use in the rehabilitation of P. cinnamomi infested areas. The relative rankings of the jarrah clonal lines by colonisation lengths were similar between branch and root inoculations. Branch inoculations are a valid option for testing resistance and susceptibility of young jarrah trees to P. cinnamomi. The pathogen was recovered on Phytophthora selective agar 3–6 months after inoculation from 50% of samples with lesions and 30% of symptomless samples in a series of growth cabinet, glasshouse and field experiments. However, up to 11% of samples with and without lesions and from which P. cinnamomi was not initially isolated contained viable pathogen after leaching the plant material in water over 9 days. This indicates that the pathogen could be present as dormant structures, such as chlamydospores, where dormancy needs to be broken for germination to occur, or fungistatic compounds in the tissue need to be removed to allow the pathogen to grow, or both. These results have important implications for disease diagnosis and management, disease-free certification and quarantine clearance. No clonal line of jarrah was found to be 100% resistant using different inoculation methods, environmental conditions and when challenged by individuals from a large range of P. cinnamomi isolates. Even the most promising RR line had individual replicates that were unable to contain lesions or died with time. This suggests that further screening work may be required using more isolates varying in their capacity to cause disease and a broader range of environmental conditions. Jarrah clonal lines that survive such rigorous screening could then be expected to survive planting out in a range of environments in the jarrah forest and rehabilitated bauxite minesites.

Thesis (Ph.D.)--Murdoch University, 2003. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 204-220.

Thesis (Ph.D.) --Murdoch University, 2004. / Thesis submitted to the Division of Science and Engineering. Includes bibliographical references (leaves 171-185).

Thesis (Ph.D.)--Murdoch University, 2006. / Thesis submitted to the Division of Science and Engineering. Includes bibliographical references (leaves 177-213)

Thesis (Ph.D.)--Murdoch University, 2008. / Thesis submitted to the Faculty of Sustainability, Environmental and Life Sciences. Includes bibliographical references (p. 98-109)

Thesis (Ph. D.)--Murdoch University, 2007. / Thesis submitted to the Faculty of Sustainability, Environmental and Life Sciences. Includes bibliographical references (leaves 147-172).

The south-west of Western Australia has a Mediterranean climate and flora endemic to this area, including the keystone species, jarrah (Eucalyptus marginata), have adapted to the droughted summer conditions. The introduction of an exotic soil borne pathogen, Phytophthora cinnamomi, has challenged the survival of this and many other species. The expectation might be that plants stressed by drought are more susceptible to disease and this study examined the development of disease caused by P. cinnamomi in E. marginata and the significance of water status to that development. Seedlings of E. marginata, clonal plants resistant to P. cinnamomi and clonal plants susceptible to P. cinnamomi, were subjected to different watering regimes in a number of field and glasshouse experiments. To determine the level of drought stress that could be imposed on container-grown E. marginata seedlings without killing them, a preliminary experiment progressively lowered the moisture levels of the substrate in their containers, until the plants reached wilting point, at which time moisture was restored to a predetermined droughted level and the process repeated. With each subsequent droughting the wilting point was lower until it was found that the seedlings could survive when only 5% of the moisture lost from container capacity to wilting point was restored. No deaths had occurred after seedlings had been maintained at this low level for 14 days (Chapter 2). Based on these findings, the level of droughting maintained in all experiments conducted under controlled glasshouse conditions was 10% restoration. After testing the appropriateness of underbark inoculation, and a zoospore inoculation method for which no wounding was necessary, a new, non-invasive stem inoculation technique was developed. Stems were moistened in a pre-treatment, then agar plugs colonized with P. cinnamomi mycelium were held against the stem with wads of wet cotton wool and bound in place with tape. This technique resulted in a high proportion of infection in E. marginata (Chapter 4) without the need for underbark inoculation or the use of zoospores (Chapter 3). It was successfully used in a large field trial in a rehabilitated bauxite mine site with 2-year-old E. marginata clonal plants, resistant to P. cinnamomi (Chapter 5). Inoculation was in late spring after the winter and spring rainfall. This timing was to allow comparison of disease development in stressed plants under normal droughted summer conditions compared with itsdevelopment in non-stressed, irrigated plants. However, two months after inoculation, the area was deluged with unseasonal and abnormally heavy summer rainfall, negating any difference in the treatments and causing an outbreak of P. cinnamomi in the soil from an adjacent infested site. This resulted in the infection and death of some noninoculated control clones. Monitoring of the site continued for twelve months and the advance of P. cinnamomi at the site was mapped. To test the effect of drought on the expression of P. cinnamomi under more controlled conditions, a series of glasshouse experiments was set up that simulated two possible summer conditions; drought or drought followed by abnormally high summer rainfall. These experiments utilised E. marginata seedlings and clonal plants, some resistant and some susceptible to P. cinnamomi. Plants were inoculated with P. cinnamomi prior to or after droughting. Results were compared to those of control plants that had not experienced water deficit. In both seedlings and clonal plants, the greatest extent of colonization was found in plants which had experienced no water deficit. These results indicated that drought stress played a role in inhibiting the in planta development of P. cinnamomi in all genotypes (Chapter 8). This finding was consistent for both clones, susceptible and resistant to P. cinnamomi. Most recoveries were made from non-stressed clonal plants, resistant to P. cinnamomi (Chapter 6) and more colonization was found in non-stressed clonal plants, susceptible to P. cinnamomi (Chapter 7), than was recorded for droughted plants. The results of the field trial showed that P. cinnamomi was not recovered from some inoculated stems, which had obvious lesions, when segments were plated onto selective agar. This led to an intensive in vitro investigation into improved methods of recovery. Dark brown exudates from some segments of inoculated stems stained the surrounding agar onto which they were plated, suggesting the presence of phenolic compounds. Recovery of the pathogen from stems increased by about 10% when segments were first soaked in distilled water to leach out the phenolic compounds, then replated onto agar. Other recovery methods were also tested, including (1) baiting with Pimelea ferruginea leaves floated on the surface of water or soil filtrate, in which the infected stem segments were immersed and (2) the application of different light and temperature regimes. It was clearly shown that exudates from infected stems of field grown E. marginata inhibited the outgrowth of P. cinnamomi onto the agar. To counter the possible toxic effect that oxidized phenolics had on the growth of the P. cinnamomi, an antioxidant was added to the agar. P. cinnamomi was grown on media whichincorporated exudates from infected stems and different concentrations of ascorbic acid, with and without adjusted pH levels. There was a pronounced pH effect, with less growth on media with lower pH and no significant increase in growth of the mycelium with increased ascorbic acid concentration on pH adjusted agar (Chapter 9). The inhibitory effect of the exudates from the stem segments led to an investigation of the possibility that, if seedlings to be planted in the rehabilitation process could be pre-treated with phenolic compounds to render them more resistant, they may have an advantage when establishing in areas where there was a potential threat of P. cinnamomi. E. marginata seeds were germinated and the seedlings grown hydroponically in a constant temperature growth room. Different concentrations of synthetic catechol, a phenolic compound naturally occurring in E. marginata, were added to the nutrient solution. Roots remained immersed in the catechol solutions for three days, before being inoculated at the root tip with zoospores of P. cinnamomi. Roots in higher concentrations of catechol were less colonized than those in lower concentrations, indicating an increased resistance to the pathogen (Chapter 10). Further work is required to determine if seedlings treated before being planted in areas threatened by an outbreak of P. cinnamomi have a greater capacity for survival, and for how long the protection persists. The improved recovery of P. cinnamomi from infected plants is important for accurate assessment of the spread of the disease in an area and for the subsequent implementation of management strategies of containment and control. An outbreak of P. cinnamomi can impact on the revegetation of rehabilitated mine sites and the aetiology of the pathogen in mine sites needs to be more fully understood. The interaction of plant defences with the invasive pathogen has been examined in a range of environments in the field, the glasshouse, in a hydroponics system and in vitro. The results indicate that summer droughting increases the resistance of E. marginata to P. cinnamomi. However, more work is required to understand the mechanisms involved. The study also indicates that clones of E. marginata, selected as resistant to P. cinnamomi, are not resistant under all conditions and that environmental interactions should be further investigated. Lastly, for effective management strategies to be implemented it is critical that the pathogen can be confidently isolated from plants. It was shown that exudates from infected hosts inhibit the recovery of P. cinnamomi. Recovery methods that can overcome these inhibitory compounds are required. The findings invite further research into the complexity of host-pathogen relationships.