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Saprophytic ability and the contribution of chlamydospores and oospores to the survival of Phytophthora cinnamomi

McCarren, Kathryn (2006) Saprophytic ability and the contribution of chlamydospores and oospores to the survival of Phytophthora cinnamomi. PhD thesis, Murdoch University.

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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 mcg 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 mcg diameter and < 20 mcg 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.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): Hardy, Giles
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