Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Phytophthora cinnamomi has been recognised as a key threatening process to Australia’s biodiversity by the Commonwealth’s Environment Protection and Biodiversity Conservation Act 1999. Despite over 80 years of extensive research, its exact mode of survival is still poorly understood. It is widely accepted that thin- and thick-walled chlamydospores are the main survival propagules while oospores are assumed to play no role in the survival of the pathogen in the Australian environment, yet evidence is limited. The saprophytic ability of the pathogen is still unresolved despite the important role this could play in the ability of the pathogen to survive in the absence of susceptible hosts. This thesis aimed to investigate chlamydospores, oospores and the saprophytic ability of P. cinnamomi to determine their contribution to survival. Phytophthora cinnamomi did not show saprophytic ability in non-sterile soils. The production of thick-walled chlamydospores and selfed oospores of P. cinnamomi in vitro was documented. Thick-walled chlamydospores were sporadically formed under sterile and non-sterile conditions in vitro but exact conditions for stimulating their formation could not be determined. The formation of thick-walled chlamydospores emerging from mycelium of similar wall thickness was observed, challenging the current knowledge of chlamydospore formation. Selfed oospores were abundant in vitro on modified Ribeiro’s minimal medium in one isolate. Three other isolates tested also produced oospores but not in large numbers. Although the selfed oospores did not germinate on a range of media, at least 16 % were found to be viable using Thiozolyl Blue Tetrazolium Bromide staining and staining of the nuclei with 4´, 6-diamidino-2-phenylindole.2HCl (DAPI). This indicated the potential of selfed oospores as survival structures and their ability to exist dormantly. The ability of phosphite to kill chlamydospores and selfed oospores was studied in vitro. Results challenged the efficacy of this chemical and revealed the necessity for further study of its effect on survival propagules of P. cinnamomi in the natural environment. Phosphite was shown to induce dormancy in thin-walled chlamydospores if present during their formation in vitro. Interestingly, dormancy was only induced by phosphite in isolates previously reported as sensitive to phosphite and not those reported as tolerant. Chlamydospores were produced uniformly across the radius of the colony on control modified Ribeiro’s minimal medium but on medium containing phosphite (40 or 100 µg ml-1), chlamydospore production was initially inhibited before being stimulated during the log phase of growth. This corresponded to a point in the colony morphology where mycelial density changed from tightly packed mycelium to sparse on medium containing phosphite. This change in morphology did not occur when the pathogen was grown on liquid media refreshed every four days, and chlamydospores were evenly distributed across the radius of these colonies. This trend was not observed in selfed oospores produced in the presence of phosphite. Selfed oospore production was found to be inhibited by phosphite at the same concentrations that stimulated chlamydospore production. Isolates of P. cinnamomi were transformed using a protoplast/ polyethylene glycol method to contain the Green Fluorescent Protein and geneticin resistance genes to aid in future studies on survival properties of the organism. Although time constraints meant the stability of the transgene could not be determined, it was effective in differentiating propagules of the transformed P. cinnamomi from spores of other microrganisms in a non-sterile environment. Two different sized chlamydospores (approximately 30 µm diameter and < 20 µm diameter) were observed in preliminary trials of transformed P. cinnamomi inoculated lupin roots floated in non-sterile soil extracts and these were easily distinguished from microbial propagules of other species. The growth and pathogenicity was reduced in two putative transformants and their ability to fluoresce declined over ten subcultures but they still remained resistant to geneticin. This study has improved our knowledge on the survival abilities of P. cinnamomi in vitro and has provided a useful tool for studying these abilities under more natural glasshouse conditions. Important implications of phosphite as a control have been raised.

Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-12-07Bitstream added on 2014-06-13T19:05:02Z : No. of bitstreams: 1 bueno_cj_dr_botfca.pdf: 2018013 bytes, checksum: b7b623474d47dc3de37a05d86cd72b0f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Os fungos fitopatogênicos habitantes de solo causam grandes perdas em culturas econômicas. Estes organismos, por produzirem estruturas de resistência na ausência de hospedeiros e/ ou condições climáticas desfavoráveis, inviabilizam o controle do patógeno. A preservação de fungos por longos períodos é importante para que pesquisas possam ser realizadas a qualquer tempo. O método de manutenção destes organismos requer que sejam conservados em baixa atividade biológica para preservar as características de esporulação e patogenicidade. O objetivo deste trabalho foi desenvolver metodologias para produzir, avaliar a sobrevivência e preservar as estruturas de resistência dos fungos Fusarium oxysporum f.sp. lycopersici raça 2, Macrophomina phaseolina, Rhizoctonia solani GA4 HGI, Sclerotium rolfsii, Sclerotinia sclerotiorum e Verticillium dahliae. O primeiro experimento foi feito no delineamento de parcelas inteiramente casualizadas em que a sobrevivência das estruturas de resistência dos seis fungos foi avaliada mensalmente por seis meses, em condições de campo e de laboratório. Para tanto, estruturas de cada fungo, produzidas através das metodologias desenvolvidas, foram enterradas no campo a 10 cm de profundidade e outras mantidas no laboratório. O objetivo deste experimento foi verificar a eficiência das metodologias para manter e também para avaliar a capacidade da sobrevivência dos patógenos, permitindo desta forma, manusear as estruturas de resistência de todos os fungos estudados. No segundo experimento, frascos de 500 mL (duas repetições/fungo) contendo estruturas de resistência, produzidas através das 2 metodologias desenvolvidas, foram mantidos em temperatura natural de laboratório, de refrigeração (º5C) e de freezer (-20ºC) para determinar a melhor condição para preservar as estruturas de cada fungo...

Phytophthora cinnamomi is an important pathogen of many plant species in natural ecosystems and horticulture industries around the world. In Western Australia, a high proportion of native plant species are susceptible to P. cinnamomi attack. Acacia pulchella, a resistant legume species native to Western Australia has been considered as a potential biological control tool against P. cinnamomi. To develop effective control methods, it is important to understand the interactions between the control agent and the different life forms of the pathogen. In this thesis the interactions are investigated between P. cinnamomi and varieties of A. pulchella which occur in jarrah (Eucalyptus marginata) forest and sand plain ecosystems. The soil inoculum of P. cinnamomi was compared under the potted plants of the three common varieties of A. pulchella, var. pulchella, var. glaberrima and var. goadbyi. These were grown in infected jarrah forest soil in the glasshouse and in vitro in a sterilised soil-less mix aseptically. Acacia urophylla (a species non suppressive towards P. cinnamomi) was also included as a control. An isolate of the most commonly found clonal lineage of P. cinnamomi in the jarrah forest, A2 type 1 was selected for use in experiments after testing showed it reliably produced zoospores and chlamydospores both axenically and in non-sterile conditions, in comparison to several other isolates. The lowest survival of P. cinnamomi inoculum was found under A. pulchella var. goadbyi plants grown both in non sterile soil and in aseptic soil-less mix. All the life forms of P. cinnamomi were affected by A. pulchella (Chapters 2, 3, 4 and 5). The soil leachates from potted plants of A. pulchella var. goadbyi reduced sporangial production (Chapter 2) and caused cytoplasm collapse of chlamydospores (Chapter 3). The confirmation was obtained that soil under A. pulchella was inhibitory to sporangial stage of P. cinnamomi and new evidence was obtained on chlamydospore inactivation. Cytoplasm collapse in the chlamydospores was observed both for chlamydospores on mycelial discs on Mira cloth exposed to the soil leachate and within infected roots buried in soils under the three varieties of A. pulchella plants. The effect was strongest under the plants of A. pulchella var. goadbyi and indicated that the chlamydospores of P. cinnamomi are unlikely to act as persistent structures under A. pulchella var. goadbyi plants. In Chapter 4, bioassays were conducted with axenically produced mycelia, chlamydospores and zoospores to test the inhibitory effect of the root exudates collected from aseptically grown A. pulchella var. goadbyi plants. The zoospores of the same isolate used in the soil leachate tests were immobilised (became sluggish and encysted) within one to two minutes. When incubated for 24 h, zoospores predominantly clumped and germ tubes were observed only from the clumped ones. Chlamydospores produced by four isolates of the common A2 type 1 strain and the only one A2 type 2 strain available at the time were tested. A higher percentage of chlamydospores collapsed and a very low percentage germinated after 24 h. Chlamydospores of all the A2 type 1 isolates were inhibited by the root exudates whilst the A2 type 2 isolate remained viable. The findings showed that the suppressive effect must be due at least in part to substances exuded by the A. pulchella plants. However, it appeared that the A2 type 1 isolates were more vulnerable to this effect than the single A2 type 2 isolate. In Chapter 5, the effect of season on sporangial suppression of P. cinnamomi was shown using field soils collected from three jarrah forest soil vegetation types and a Banksia woodland on Bassendean sand, collected in winter and summer. The effect of age of A. pulchella plants was demonstrated using the soils collected from rehabilitated bauxite mine pits. In all the locations soils were collected under A. pulchella plants and 5 m away from the nearest A. pulchella. An effect of soil type was evident as whilst the soil leachates made from the three lateritic jarrah forest soil types where A. pulchella is common in the understorey were suppressive to the sporangial stage of P. cinnamomi, this effect was not evident in the Bassendean sand under A. pulchella. A. pulchella soils collected in winter were less suppressive towards sporangial production than soils collected in summer. An effect of plant age was demonstrated as soil leachates from four year-old A. pulchella stands in rehabilitated bauxite mine sites were more suppressive for sporangia than leachates from one year-old stands. Further information on the behaviour of the pathogen in soil and in potting mix with and without A. pulchella was obtained by infecting lupin radicles with an isolate of each A2 type, 1 and 2 strains of P. cinnamomi and burying them in the soil under the three varieties of A. pulchella plants. After a week, the chlamydospores were mostly collapsed and hyphae deteriorated. Oospores were observed and in significant numbers under the potted plants of A. pulchella var. glaberrima. Isolates of all three clonal lineages of P. cinnamomi found in Australian soil were tested for the ability to produce oospores. Two isolates of the A1 and A2 type 2 and three isolates of the common A2 type 1 were screened. The two isozyme types of the A2 clonal lineage isolated in Australia varied in ability to self and produce oospores in planta in several soils from the jarrah forest. The isozyme type 2 of the A2 clonal lineage of P. cinnamomi produced oospores under these experimental conditions. This stimulation was not effective for most of the tested isolates of the A2 type 1 and the A1 clonal lineage. The in planta oospores were viable but dormant and the oogonial-antheridial associations were amphigynous both in vitro and in vivo. For the first time it was established that, the stimulus for selfing and oospore formation in the A2 type 2 of P. cinnamomi is available in some jarrah forest soils, with and without A. pulchella and also in the potting mix used. This raises important questions for the management of the pathogen. Several factors were identified as potential stimuli for selfing. Among them, soil nutrient levels and essentially enhanced sulphur presence were found important. Temperature also played a key role. Oospores were produced abundantly at 21 – 25 ºC but not over 28 ºC. The biology of P. cinnamomi has been studied for several decades but some important aspects remain un-researched. This thesis pioneers research into the in planta selfing aspect of the pathogen in soil. It also improved the understanding of the interactions between P. cinnamomi and A. pulchella which to some extent supports use of A. pulchella as a biological control tool against P. cinnamomi. However, attention is drawn to the natural mechanisms of this complex pathogen to survive in planta by producing oospores, the most persistent form of its life cycle.

Orientador: Nilson Luiz de Souza / Banca: Edson Luiz Furtado / Banca: Yodiro Masuda / Banca: Raquel Ghini / Banca: Walmir Contra de Jesus Junior / Resumo: Os fungos fitopatogênicos habitantes de solo causam grandes perdas em culturas econômicas. Estes organismos, por produzirem estruturas de resistência na ausência de hospedeiros e/ ou condições climáticas desfavoráveis, inviabilizam o controle do patógeno. A preservação de fungos por longos períodos é importante para que pesquisas possam ser realizadas a qualquer tempo. O método de manutenção destes organismos requer que sejam conservados em baixa atividade biológica para preservar as características de esporulação e patogenicidade. O objetivo deste trabalho foi desenvolver metodologias para produzir, avaliar a sobrevivência e preservar as estruturas de resistência dos fungos Fusarium oxysporum f.sp. lycopersici raça 2, Macrophomina phaseolina, Rhizoctonia solani GA4 HGI, Sclerotium rolfsii, Sclerotinia sclerotiorum e Verticillium dahliae. O primeiro experimento foi feito no delineamento de parcelas inteiramente casualizadas em que a sobrevivência das estruturas de resistência dos seis fungos foi avaliada mensalmente por seis meses, em condições de campo e de laboratório. Para tanto, estruturas de cada fungo, produzidas através das metodologias desenvolvidas, foram enterradas no campo a 10 cm de profundidade e outras mantidas no laboratório. O objetivo deste experimento foi verificar a eficiência das metodologias para manter e também para avaliar a capacidade da sobrevivência dos patógenos, permitindo desta forma, manusear as estruturas de resistência de todos os fungos estudados. No segundo experimento, frascos de 500 mL (duas repetições/fungo) contendo estruturas de resistência, produzidas através das 2 metodologias desenvolvidas, foram mantidos em temperatura natural de laboratório, de refrigeração (º5C) e de freezer (-20ºC) para determinar a melhor condição para preservar as estruturas de cada fungo...(Resumo completo, clicar acesso eletrônico abaixo) / Soilborne phytopathogenic fungi cause great losses in economical cultures. These organisms produce resistance structures in the absence of hosts and / or unfavorable climatic conditions. These structures, when presents, make unfeasible the pathogen control. The preservation of fungi for long periods is important to make possible researches the any time. The method of maintenance of these organisms requests that are conserved in low biological activity to preserve the sporulate characteristics and pathogenicity. The objective of this work was to develop methodologies to produce and to evaluate the survival and also to preserve the resistance structures of Fusarium oxysporum f.sp. lycopersici race 2, Macrophomina phaseolina, Rhizoctonia solani AG4 HGI, Sclerotium rolfsii, Sclerotinia sclerotiorum and Verticillium dahliae. The first experiment was carried out in a randomized plots design where the survival of the resistance structures of the six fungi was monthly evaluated for six months under field and laboratory conditions. The structures of each fungus, produced through the developed methodologies, were buried in the field at 10 cm of depth and another maintained at the laboratory. The objective of this experiment was to verify the efficiency of the methodologies to maintain and also to evaluate the survival capacity of the pathogens in order to set methodologies to handle the resistance structures of those fungi. In the second experiment 500 mL flasks (two repetitions/fungus) containing resistance structure produced through developed methodologies were maintained at room, refrigerator and freezer temperature to determine the best condition to preserve the structures those pathogens. Monthly and for a period of 4 one year the survival and the vigour of each pathogen were evaluated on their specific medium...(Complete abstract click electronic access below) / Doutor

The movement of the exotic and destructive plant pathogen Phytophthora ramorum into unquarantined areas via the plant nursery trade provides a potential outlet for transmission into eastern United States forests. A two-year survey of Phytophthora species in a forest adjacent to an ornamental plant nursery in Mississippi isolated P. ramorum 20 times from water and once from vegetation, with an additional detection of 14 Phytophthora species and one provisional species. Isolates were recovered from soil, water, and vegetation using baiting and filtering techniques, and verified by their DNA through Polymerase Chain Reaction (PCR) followed by genomic sequencing. This study confirms the ability of P. ramorum to sustain itself in Mississippi, although disease progression appears to be inhibited by the relatively small window of favorable environmental conditions.