9/05/2013 Dealing with Phytophthoras – our story in Western Australia Overview of Talk • Overview of species found globally • Sampling techniques and ‘tweaking’ to improve isolation success rates • Examples of new Phytophthora species in Australia (when you look). • New biological /ecological surprises with regards pathogen survival, pathology and hosts • Eradication and containment methods Giles Hardy, • Phosphite –pros and cons Treena Burgess, Bill Dunstan, Trudy • Monitoring tree health using remote sensing tools Paap, Nari Williams, Peter Scott, Thomas Jung, Michael Crone (and others) • Currently ~123 Phytophthora species described worldwide Sampling symptomatic plants for the presence of • Estimated another 100‐300 species will be described from Phytophthora woody plants Number of described Phytophthora species within each Clade Necrotic lesion below bark in cambium and into wood of a jarrah before and after the year 2000 Soil / Rhizosphere soil sieving BAITING for Phytophthora species • Young Leaves (many species good) •Cotyledons Sieve a number of composite samples per location •2‐3 day‐old Lupinus angustifolious CRITICAL TO: • plate out lesions onto selective agar immediately they become obvious • to avoid opportunistic Pythium spp. Remove as much soil as possible since P. cinnamomi (and other species) are generally poor saprotrophs If a ‘negative’ result dry the soils – rewet and rebait This approach increases positive recoveries substantially, Note ‘Fly wire mesh’ to Allows for the combination of composite samples ensure floating OM does not and reduces the amount (weight) of soil being carried touch the baits To the laboratory and eventually autoclaved! Start plating out immediately you see lesions 1 9/05/2013 Phytophthora species in natural ecosystems in WA •Until recently identified only by morphological features P. citricola P. m e g as p e rm a P. cryptogea Clade 6 • Obtained by ‘Fishing for Phytophthora’ –yet to P. drechsleri determine its role as a phytopathogen P. nicotianae P. boehmeriae •Other species are found in nurseries and in horticulture •Over the past 5 years we have been conducting a molecular re- evaluation of Phytophthora spp. in natural ecosystems in WA •Nearly 1000 non-P. cinnamomi isolates so far examined from VHS (Dept Environment and Conservation) collection (~30 years) •Initially sequenced ITS region and constructed molecular Plate ‘TINY’ pieces and phylogeny; other gene regions sequenced for species descriptions lots of them (2 x 2 mm)!! Use many different baits 100 Phytophthora nicotianae 1 67 Phytophthora citricola Clade 2 86 92 Phytophthora aff. arenaria 100 Phytophthora alticola 4 Phytophthora pini 99 88 Phytophthora arenaria 100 Phytophthora palmivora Phytophthora plurivora 88 Phytophthora citricola 100 91 52 Phytophthora plurivora 2 100 Phytophthora multivora Phytophthora multivora 100 Phytophthora elongata 92 Phytophthora aff. elongata 91 66 Phytophthora rosacearum 95 68 Phytophthora aff. rosacearum 100 90 Phytophthora taxon kwongan 98 Phytophthora taxon cooljarloo 80 66 Phytophthora humicola 86 Phytophthora aff. humicola 60 86 100 Phytophthora inundata 100 Phytophthora taxon personii 6 83 Phytophthora gibbosa Phytophthora gregata 100 57 Phytophthora fluvialis 100 Phytophthora amnicola 83 100 Phytophthora litoralis 100 65 95 Phytophthora thermophila 73 100 Phytophthora megasperma 100 88 Phytophthora taxon PgChlamydo 100 100 Phytophthora lacustris 100 Phytophthora asparagi 100 100 Phytophthora taxon niederhauserii 100 100 Phytophthora cinnamomi var. parvispora 7 100 Phytophthora cinnamomi 100 Phytophthora cryptogea 8 96 100 Phytophthora captiosa 100 94 Phytophthora fallax 9 100 100 Phytophthora constricta 100 Phytophthora boehmeriae 10 5 changes Previously part of P. c i t r i c o l a complex. Large host range. Eucalyptus gomphocephala, Phytophthora multivora P. frigida B. attenuata, B. littoralis, B. menziesii, B. prionotes, P. bicheria Conospermum sp., Leucopogon verticillatus, P. elongata Xanthorrhoea gracilis, Podocarpus drouyniana, Patersonia sp., Bossiaea sp., P. aff. elongata Gastrolobium spinosum, Pinus radiata, E. Clade 2 marginata, Ficus sp. (> 26 species) Previously recorded as P. citricola (Psp2) Hosts associated with: E. marginata, Corymbia calophylla, Andersonia sp., Patersonia xanthia, Banksia grandis, Xanthorrhoes preissii, X. gracilis, Leucopogon propinquus Dryandra squarrosa Only on lateritic soils in WA and in Victoria on sandy soils 2 9/05/2013 Clade 4 Phytophthora constricta Phytophthora quercetorum Phytophthora aff. captiosa Phytophthora aff. arenaria Phytophthora captiosa Phytophthora alticola Phytophthora fallax Phytophthora arenaria Phytophthora quercetorum Phytophthora aff. arenaria Phytophthora alticola Phytophthora arenaria P. multivora P. constricta P. inundata P. cryptogea P. nicotianae P. arenaria P. constricta IVAL SURV P. multivora P. elongata Pathogenicity- Endemic pathogens? host distribution Mortality of B. attenuata induced by P. constricta and P. arenaria was P. humicola Citrus, Phaseolus Taiwan, Japan dependent:- • on a flooding stimulus and showed a variation between isolates of 10– P. aff humicola Casuarina obesa Western Australia 25%. P. inundata numerous Western Australia, Europe In contrast, the introduced pathogen P. cinnamomi caused 100% P. taxon personii Nicotiana, Grevillea Western Australia, USA mortality of seedlings and did not require a flooding stimulus to initiate death. P. gemini Zostera marina the Netherlands P. cinnamomi forms a visible and indiscriminate path of destruction P. taxon cyperaceae Sedge Western Australia through entire plant communities, P. constricta and P. arenaria have a more limited impact, selectively killing species belonging predominantly P. rosacearum Malus, Prunus, Pinus, Western Australia, USA Xanthorrhoea to the family Proteaceae. P. aff. rosacearum native vegetation Western Australia The incidence of P. constricta or P. arenaria is usually episodic following extreme rainfall events, which is congruent with the results of the soil P. taxon kwongan Banksia, Xanthorrhoea Western Australia infestation trial, where flooding was required to cause disease. P. taxon cooljarloo Hibbertia Western Australia • This supports the hypothesis of host–pathogen coevolution. P. taxon walnut Juglans USA (California) • Are these species endemic to Western Australia? 3 9/05/2013 Significance of new Phytophthora species DO THEY POSE A THREAT TO BIODIVERSITY? •P. multivora and not P. cinnamomi is implicated in tuart decline •P. multivora is the common cause of basal cankers in Banksia spp. and Agonis flexuosa •P. multivora is the most commonly isolated species from dying plants in the urban and peri‐ urban areas P. aff. humicola P. aff. cyperaceae •P. arenaria is the most common species causing death of Banksia and other species in arid environments (ie. northern sand plain) •P. constricta is the most common species causing death of Banksia in southern heathlands •P. elongata causes episodic collapse of jarrah and marri rehabilitation on bauxite minesites P. rosacearum •P. aff. arenaria has caused the death of thousands of seedlings in a oil mallee (Eucalyptus sp.) nursery (DAMPING‐OFF PATHOGEN, BUT ALSO REDUCES GROWTH OF OLDER PLANTS) P. aff. Rosacearum clade 6 •P. aff humicola regularly isolated from dying Casuarina P. taxon kwongan clade 6 • Hybrids have huge potential to be problematic (ESPECIALLY IN NURSERY ENVIRONMENTS) P. taxon cooljarloo Phytophthora spp. predisposing to other pathogens?? Marri Canker We isolate P. cinnamomi, P. multivora, P. elongata and others from trees with cankers Pathogenicity trial with P. arenaria and P. aff. arenaria on oil mallee trees Control left hand‐side Quambalaria Inoculated right hand‐side coyrecup – native MARRI CANKERS Persistence of Phytophthora cinnamomi on black gravel graveyard Sites Michael Crone Trachymene pilosa Crassula closiana Chamaescilla corymbosa Stylidium diuroides 4 9/05/2013 Annuals and herbaceous perennials as new Mode of growth in annual and herbaceous hosts of Phytophthora cinnamomi perennial plant species (Biotroph/Endophyte?) * 15 of 19 annual and herbaceous perennial plant species were found to be hosts of P. cinnamomi •67% were asymptomatic hosts didespite bibeing ifinfecte d and extensively colonised by P. cinnamomi •What survival propagules are produced in these symptomless hosts?? Haustorium Production of ‘selfed’ oospores (3‐400/m2) Oospores – Proof of identity Amphigynous 1. P. cinnamomi specific 2. Sequencing of oospore isolate oospore molecular staining (DNA analysis) Characteristic hyphae Fluorescent in situ hybridisation (FISH) Thick‐walled chlamydospores Stromata –A survival structure for P. cinnamomi 2 1 Germination of thick walled chlamydospore with multiple germ tubes. 3 4 5 9/05/2013 Stromata produce oospores and chlamydospores Lignitubers Stromata Eradication –Treatment approach Chemical Attack Vegetation destruction Field Operations: Cape Riche Results: Cape Riche Untreated (0.5m) Untreated (2.5 m) Treated (0.5m) Treated (2.5m) Calculated probability that we failed to detect P.c in treated plots post treatment was 1.6 x 10-12 - 8.7x10-9 6 9/05/2013 Can Phytophthora cinnamomi survive through Results: 2.5 years of monitoring two summers without any host material? Number of recoveries SITE 1 SITE 2 Total isolations Total isolations Control (1C) Eradication (1E) Control (2C) Eradication (2E) PRIOR TREATMENT 10 11 10 15 AFTER TREATMENT 1 year 7 5 10 6 2 years 12 3 11 3 2.5 years (Autumn 2012) 3 0 2 0 Major observations Management implications