Phylogenetic relationships of Phytophthora species based on ribosomal ITS I DNA sequence analysis with emphasis on Waterhouse groups V and VI

https://researchrepository.murdoch.edu.au/870/1/phenotypic_variation.pdf

Phenotypic variation in a clonal lineage of two Phytophthora cinnamomi populations from Western Australia

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.

/pdf/saprophytic-ability-and-the-contribution-of-chlamydospores-2urhnwoe5j.pdf

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

Four isolates of Phytophthora cinnamomi were able to produce selfed oospores when cultured in vitro on modified Ribeiro's minimal medium. Phosphite inhibited the production of selfed oospores at 100 μg phosphite/ml, lower concentrations had no effect. Selfed oospores were considered dormant as 16-47% of selfed oospores were found viable using viability staining techniques such as thiazolyl blue tetrazolium bromide and 4′,6-diamidino-2- phenylindole•2HCl yet they did not germinate on a range of media. Phosphite up to 100 μg/ml did not change the proportion of viable selfed oospores. This study shows that phosphite does not affect the production of selfed oospores.

Phosphite impact on the in vitro production and viability of selfed oospores by Phytophthora cinnamomi

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 degrees C, it was found that the method of inoculation did not produce comparable results, particularly at 25 and 30 degrees 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 degrees 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 degrees 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.

Phenotypic variation of two localised populations of Phytophthora cinnamomi from Western Australia and how they impact on Eucalyptus marginata resistance

The position of the antheridium on the oogonium of Phytophthora boehmeriae was greatly affected by culture media. Agar medium containing lecithin and basal salts and corn grain agar favored the production of paragynous antheridia, while corn meal agar, lima bean agar, vegetable juice agar, and tomato agar were conducive to formation of amphigynous antheridia. The effect of culture media on antheridial configuration was found to be due to nutrient concentration. A high concentration of nutrients in the media was favored the formation of amphigynous antheridia, and a low concentration of nutrients favored paragynous antheridia.Key words: antheridial configuration, oogonium, nutrients, Phytophthora boehmeriae.

Z M Gao

Papers

Effect of culture media on antheridial configuration in Phytophthora boehmeriae