From ‘then to now’ – Phytophthora science and management in Western Australia • Work from many Postdocs, PhD and Honours students Overview of Talk Disease development and long-term survival of Phytophthora cinnamomi and can we eradicate it? • Overview of Phytophthora impact in Australia. • Alcoa’s mining and restoration activities. • Emphasis on the biology, ecology, pathology and control of Phytophthora cinnamomi in the jarrah forest. • Will not talk about phosphite work, plantation work, or climate change and woodland and forest health research. Phytophthora Taxonomy • Phytophthora species are ‘Water Moulds’ or Oomycetes they are not True Fungi • More closely related to brown algae than fungi • Filamentous Protists - Kingdom Chromista Currently, approximately 150 Phytophthora species described worldwide Estimated another 100-300 species will be described from trees P. cinnamomi isolations and broad climatic envelope of P. cinnamomi susceptibility in Australia (O’Gara et al. 2005b) Eucalyptus marginata (jarrah) forest Kwongan heaths Severely infested ‘Black gravel’ site Banksia Woodland on Bassendean Sands South of Perth Fitzgerald River National Park Loss of susceptible species Along creek lines Healthy montane heath in Stirling Ranges Diseased health in Stirling Ranges Impact of Phytophthora cinnamomi on plant species in Western Australia Direct Impacts • Out of 5710 described species in the South-West Botanical Province • 2285 species susceptible (40%) • 800 highly susceptible (14%) Indirect Impacts Indirect Impacts • Loss of biomass • Increased exposure • Loss of litter •Loss of pollinators • Loss of refugia for fauna •Loss of nesting sites • Loss of food resources Alcoa Australia To understand the biology, ecology, pathology and control of P. cinnamomi before, during and post-mining Sporangia and zoospores Zoospores Chlamydospore C Oospore • Widely assumed chlamydospores are the main survival propagules • Oospores assumed to play no role in survival • Saprophytic ability unresolved in terms of survival Jarrah forest is a mosaic Podocarpus drouyniana Xanthorrhoea preissii Xanthorrhoea gracilis PatersoniaPatersoniasp. sp. Banksia grandis First indication of presence of P. cinnamomi is the death of susceptible ‘indicator’ species from the families Proteaceae, Epacridaceae, Dilleniaceae, Xanthorrhoeaceae, Fabaceae Phytophthora Management Map Disease-free Uninterpretable Phytophthora infested Green = Dieback free; Purple = Uninterpretable; Red = Infested Once P. cinnamomi has been confirmed in the landscape the areas to be ‘managed’ are marked on the ground. Uninfested Infested Usually ‘blazed’ 15-20m from actual ‘dieback front’ INFESTED Front Barrier Jarrah forest being cleared prior Removing topsoil & overburden to mining Caprock Active minesite Ore has been blasted and ready for removal Removing bauxite to crusher Pc free topsoil pile Spreading topsoil Deep ripping and contouring One year-old restored minepit P. cinnamomi infested two year old restored pit Ponding Necrotic lignotuber in Marri Bag trial to look at infection and disease development over time • Never isolated P. cinnamomi from roots – even when plugs touching roots • Disease only developed in collars where ponding occurred in a few trees • Always isolated from the inoculum plugs even at depth • Only isolated from collars of trees that had been ‘ponded’ Zoospores added to the cups Encysted and germinating zoospores Zoospores attracted to the stem leaf scar (SLS) of jarrah stem. (b) Enlargement of SLS showing uniform orientation of zoospores towards the host (c) Zoospores 500 micron 200 micron Axillary shoot (AS) emerging through periderm (P) above a stem-leaf-scar. (d) Enlargement of AS showing zoospores on and between the new leaves Low oxygen levels on disease development in resistant and susceptible clones of jarrah 1. Normal Oxygen (8mg/L O2) 2. Hypoxia (6 days at 2mg/L O2) 3. Anoxic acclimatization 2 days at 2mg, 2 days at 1mg, 6 hours at 0.05 mg/L O2 4. 6 h of anoxia at <0.05 mg/L O2 Resistant clone Susceptible clone Natural vs artificially produced inoculum Long-term survival using ‘natural’ inoculum Placed into: - Soil maintained at container capacity - Soil at container capacity and allowed to dry slowly - Harvested over 450 days - Selective agar or baited - Histological studies Survival and types of inoculum Container capacity Allowed to dry out over time Artificial inoculum Natural Inoculum Comparison between autoclaved and naturally infected Banksia grandis stems – showing thin-walled and thick walled chlamydospores Standard errors of the mean in parentheses Dormancy of thick-walled chlamydospores?? % recovery of P. cinnamomi over 34 months from B. grandis trunk and root samples at 10 and 30cm above and below ground Data show recovery from bark and wood combined for trees that died in (a) summer of 2000 (10 months after inoculation) or (b) summer of 2001 (22 months after inoculation). Saprophytic ability 100%, 85% and 50% container capacity • Sterile vs non-sterile soils • Different isolates • Different soil matric potentials • Different temperatures P. cinnamomi unable to grow saprophytically in non-sterile soil. It could not colonise new lupin baits placed in the soil Thick-walled chlamydospores of isolates of Phytophthora cinnamomi produced after 4 weeks on modified Ribeiro’s minimal medium. (Bar = 30 µm). Formed in the presence of a minimal medium over 4 weeks, in the presence of A. pulchella and Banksia grandis leachates in liquid culture and in lupin root in non sterile soil leachate Many chlamydospores were smaller than 30 µm in diameter. Different P. cinnamomi isolates differed in ability to produce thick- walled chlamydospores Chlamydospores of Phytophthora cinnamomi within inoculated lupin roots in jarrah vegetation Q red loam and potting mix Bars = 30 µm Green Fluorescent Protein ERADICATION AND CONTAINMENT Herbicides Treated (2.5m) Cape Arid N.P. (Telegraph Track) Vegetation: scrub-heath (Myrtaceae, Proteaceae, Fabaceae, Ericaceae, etc.) Soils: White siliceous sand with ‘coffee rock’ (podosol/podzol?) Infestation: ca. 500 m2 Treated area: ca. 2500 m2 Cape Arid - Treatments Herbicides (glyphosate + metsulfuron) Fumigant: MITC (Metham + Basamid) Treatments very effective against: Cape Arid - Results • P. cinnamomi No recoveries of • Seed banks- therefore P. cinnamomi at 12 and potential hosts of P. 27 months after cinnamomi completion of treatments. Follow up treatments for few surviving seedlings Persistence of Phytophthora cinnamomi on black gravel graveyard Sites Michael Crone Trachymene Crassula pilosa closiana Chamaescilla corymbosa Stylidium diuroides Annuals and herbaceous perennials as new hosts of Phytophthora cinnamomi * 15 of 19 annual and herbaceous perennial plant species were found to be hosts of P. cinnamomi •67% were asymptomatic hosts despite being infected and extensively colonised by P. cinnamomi •What survival propagules are produced in these symptomless hosts?? Mode of growth in annual and herbaceous perennial plant species (Biotroph/Endophyte?) Haustorium Production of ‘selfed’ oospores (3-400 mm-2) Amphigynous oospore Characteristic hyphae Oospores – Proof of identity Fluorescent in situ hybridisation (FISH) 1. P. cinnamomi specific 2. Sequencing of oospore isolate staining & 2. FISH (in situ fluorescent hybridisation Thick-walled chlamydospores Germination of thick walled chlamydospore with multiple germ tubes. Stromata – A survival structure for P. cinnamomi 2 1 3 4 Stromata Stromata produce oospores and chlamydospores Jarrah site - Eradication of Phytophthora cinnamomi Jarrah forest - results SITE 1 SITE 2 Total isolations Total isolations Eradication Eradication Control (1C) Control (2C) (1E) (2E) PRIOR TO 10 11 10 15 TREATMENT AFTER TREATMENT 1 year 7 5 10 6 2 years 12 3 11 3 2.5 years (Autumn 2012) 3 0 2 0 New information from the study of *Annual and herbaceousjarrah perennials sites are hosts of P. cinnamomi but most do not show symptoms *P. cinnamomi can grow as a biotroph/endophyte as well as a necrotroph depending on its hosts *Selfed oospores produced in high numbers *Stromata were observed for the first time in P. cinnamomi *Thick walled chlamydospores are also abundant Possible to eradicate from a site just through host removal Mining operations Operational sites: roads, bunds & sumps Operational sites: stockpiles Approaches to eradication •Host removal: herbicide treatment of all plants (Resistant, Susceptible, Tolerant) •Direct attack: fumigants (MS, CP/1,3D, EDN..) •Induction of spore germination Experimental 100 90 80 70 60 Pc not recovered 50 % % samples of Pc recovered 40 % Samples of% 30 20 10 0 road, 8 months road, 12 months lab, original inoculum (12 Inoculum source and assessment time months) Inoculum source and assessment time Conclusions • Do not believe the literature until you have read the seminal papers – ‘ghostly whispers’ • Differences between ‘natural’ inoculum and ‘artificial’ inoculum • P. cinnamomi does not ‘need’ to survive in absence of susceptible hosts for long periods of time • New survival structures still to be found? • Possible to eradicate the pathogen Thank you Pabelup –.