Overview of Talk. Dealing with Phytophthoras our story in Western Australia 9/05/2013. Sampling symptomatic plants for the presence of Phytophthora

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1 Dealing with Phytophthoras our story in Western Australia Giles Hardy, Treena Burgess, Bill Dunstan, Trudy Paap, Nari Williams, Peter Scott, Thomas Jung, Michael Crone (and others) 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 Phosphite pros and cons Monitoring tree health using remote sensing tools Currently ~123 Phytophthora species described worldwide Estimated another 300 species will be described from woody plants Sampling symptomatic plants for the presence of Phytophthora Number of described Phytophthora species within each Clade before and after the year 2000 Necrotic lesion below bark in cambium and into wood of a jarrah Soil / Rhizosphere soil sieving Sieve a number of composite samples per location Remove as much soil as possible since P. cinnamomi (and other species) are generally poor saprotrophs BAITING for Phytophthora species Young Leaves (many species good) Cotyledons 2 3 day old Lupinus angustifolious CRITICAL TO: plate out lesions onto selective agar immediately they become obvious to avoid opportunistic Pythium spp. If a negative result dry the soils rewet and rebait This approach increases positive recoveries substantially, Allows for the combination of composite samples and reduces the amount (weight) of soil being carried To the laboratory and eventually autoclaved! Note Fly wire mesh to ensure floating OM does not touch the baits Start plating out immediately you see lesions 1

2 Obtained by Fishing for Phytophthora yet to determine its role as a phytopathogen Clade 6 Phytophthora species in natural ecosystems in WA Until recently identified only by morphological features P. citricola P. megasperma P. cryptogea P. drechsleri P. nicotianae P. boehmeriae Use many different baits Plate TINY pieces and lots of them (2 x 2 mm)!! Other species are found in nurseries and in horticulture Over the past 5 years we have been conducting a molecular reevaluation of Phytophthora spp. in natural ecosystems in WA Nearly 0 non-p. cinnamomi isolates so far examined from VHS (Dept Environment and Conservation) collection (~30 years) Initially sequenced ITS region and constructed molecular phylogeny; other gene regions sequenced for species descriptions Phytophthora nicotianae 67 Phytophthora aff. arenaria 92 Phytophthora alticola Phytophthora arenaria Phytophthora palmivora 88 Phytophthora citricola 91 Phytophthora plurivora Phytophthora multivora Phytophthora citricola Phytophthora pini Phytophthora plurivora Phytophthora multivora Clade 2 60 Phytophthora elongata 92 Phytophthora aff. elongata Phytophthora rosacearum Phytophthora aff. rosacearum 90 Phytophthora taxon kwongan 98 Phytophthora taxon cooljarloo Phytophthora humicola 86 Phytophthora aff. humicola 86 Phytophthora inundata Phytophthora taxon personii 83 Phytophthora gibbosa Phytophthora gregata Phytophthora fluvialis 57 Phytophthora amnicola 83 Phytophthora litoralis Phytophthora thermophila 73 Phytophthora megasperma 6 88 Phytophthora taxon PgChlamydo Phytophthora lacustris Phytophthora asparagi 5 changes Phytophthora taxon niederhauserii Phytophthora cinnamomi var. parvispora Phytophthora cinnamomi Phytophthora cryptogea 96 Phytophthora captiosa 94 Phytophthora fallax Phytophthora constricta Phytophthora boehmeriae Previously part of P. citricola complex. Large host range. Eucalyptus gomphocephala, B. attenuata, B. littoralis, B. menziesii, B. prionotes, Conospermum sp., Leucopogon verticillatus, Xanthorrhoea gracilis, Podocarpus drouyniana, Patersonia sp., Bossiaea sp., Gastrolobium spinosum, Pinus radiata, E. marginata, Ficus sp. (> 26 species) Phytophthora multivora P. frigida P. bicheria P. elongata P. aff. elongata Clade 2 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

3 Phytophthora quercetorum Phytophthora aff. arenaria Phytophthora alticola Phytophthora arenaria Clade 4 Phytophthora constricta Phytophthora aff. captiosa Phytophthora captiosa Phytophthora fallax Phytophthora quercetorum Phytophthora aff. arenaria Phytophthora alticola Phytophthora arenaria P. multivora P. constricta P. inundata P. cryptogea P. nicotianae IVAL SURV P. arenaria P. constricta P. multivora P. elongata Pathogenicity- Endemic pathogens? Mortality of B. attenuata induced by P. constricta and P. arenaria was dependent:- on a flooding stimulus and showed a variation between isolates of 10 25%. In contrast, the introduced pathogen P. cinnamomi caused % mortality of seedlings and did not require a flooding stimulus to initiate death. P. cinnamomi forms a visible and indiscriminate path of destruction through entire plant communities, P. constricta and P. arenaria have a more limited impact, selectively killing species belonging predominantly to the family Proteaceae. The incidence of P. constricta or P. arenaria is usually episodic following extreme rainfall events, which is congruent with the results of the soil infestation trial, where flooding was required to cause disease. This supports the hypothesis of host pathogen coevolution. Are these species endemic to Western Australia? host distribution P. humicola Citrus, Phaseolus Taiwan, Japan P. aff humicola Casuarina obesa Western Australia P. inundata numerous Western Australia, Europe P. taxon personii Nicotiana, Grevillea Western Australia, USA P. gemini Zostera marina the Netherlands P. taxon cyperaceae Sedge Western Australia P. rosacearum Malus, Prunus, Pinus, Western Australia, USA Xanthorrhoea P. aff. rosacearum native vegetation Western Australia P. taxon kwongan Banksia, Xanthorrhoea Western Australia P. taxon cooljarloo Hibbertia Western Australia P. taxon walnut Juglans USA (California) 3

4 Significance of new Phytophthora species P. aff. humicola P. aff. cyperaceae P. rosacearum P. aff. Rosacearum clade 6 P. taxon kwongan clade 6 P. taxon cooljarloo 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 periurban areas 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. 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 humicola regularly isolated from dying Casuarina Hybrids have huge potential to be problematic (ESPECIALLY IN NURSERY ENVIRONMENTS) 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 Inoculated right hand side Quambalaria coyrecup native MARRI CANKERS Persistence of Phytophthora cinnamomi on black gravel graveyard Sites Michael Crone Trachymene pilosa Crassulaclosiana Chamaescilla corymbosa Stylidium diuroides 4

5 Annuals and herbaceous perennials as new hosts of Phytophthora cinnamomi Mode of growth in annual and herbaceous 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 despite being infected 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 oospore Characteristic hyphae 1. P. cinnamomi specific 2. Sequencing of oospore isolate molecular staining (DNA analysis) Fluorescent in situ hybridisation (FISH) Thick walled chlamydospores Stromata A survival structure for P. cinnamomi 1 2 Germination of thick walled chlamydospore with multiple germ tubes

6 Stromata produce oospores and chlamydospores Lignitubers Stromata Eradication Treatment approach Vegetation destruction Chemical Attack 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 x10-9 6

7 Can Phytophthora cinnamomi survive through two summers without any host material? Results: 2.5 years of monitoring Number of recoveries SITE 1 SITE 2 Total isolations Total isolations Control (1C) Eradication (1E) Control (2C) Eradication (2E) PRIOR TREATMENT AFTER TREATMENT 1 year years years (Autumn 2012) Major observations Many annual and herbaceous perennials are symptomless hosts of P. cinnamomi Haustoria indicating biotrophic mode Ability to grow as a biotroph without moving into a necrotrophic mode. Potential endophytic mode in some hosts Numerous selfed oospores produced in some hosts Stromata common: used as a nutrient springboard for oospore and chlamydospore production. Extremely plastic and versatile pathogen Management implications P. cinnamomi may be present in an area with no susceptible woody species, surviving in annual and herbaceous perennials It is possible to eradicate P. cinnamomi from black gravel sites by removing all living vegetation including annuals and herbaceous perennials Eradication may take at least years, so all dormant structures have germinated and died in absence of host plants. After this time revegetation may be undertaken in disease free conditions. Phosphite and its potential to increase Phosphorous levels in soil over time. Work by Idriss Ahmedi Natural bush P. c infested and P.c free treated with phosphite annually over 14 years Soil plant available P (PO 4 3 ) as affected by Phi accumulation. Upper horizon ( )was significantly different from lower horizon ( ). Soil total P as affected by Phi accumulation there was no significant difference upper ( )and lower horizons ( ). 7

8 Banksia attenuata mature ( ) and senesced ( ) leaves had significantly higher total P levels Phosphite treated P. c plots Phosphite treated P. c free plots phosphorus (mg/g) Mean total Non-phosphite sprayed and P. cinnamomifree Treatments Phosphite sprayed and P. cinnamomi-free Similar trends seen for Adenanthos cuneatus, Banksia coccinea and Jacksonia spinosa Non phosphite P. c plots Non phosphite P. c free plots Species diversity as affected by PC and Phi interaction in GRNP (nmds plot) 70. Phosphite trial liquid injection vs powder capsule implants Mean canopy closure (%) PHI-PC- PHI-PC+ PHI+PC+ PHI+PC- Treatments Control Mixed Phosphate Phosphite Phosphite nutrient + nutrients implants liquid Control Mixed nutrient Phosphate Phosphite Phosphite + nutrients implants liquid Mean (%) canopy cover and accompanying hemispherical photographs of the treatment sites in GRNP where a = PHI PC ; b= PHI PC+; c = PHI+PC+; and, d= PHI+PC. The bars represent the standard errors of the means where N = 10. Results show that both phosphite liquid and implants are effective at controlling lesion extension in B. grandis and E. marginata, caused by P. cinnamomi. Phosphate and mixed nutrient treatments may help control Phytophthora infections by improving plant health. Phosphite implants Composition per 1.0 g capsule Capsule Constituents Composition % by weight Dose (mg/10 cm trunk circumference) Phosphite Phosphate 4 40 Soluble potash Phosphite application liquid/soluble implant pros and cons Control Compared to phosphite liquid, phosphite implants: are less labour intensive (more user friendly); Liquid Phi require less training; require less specialized equipment; Phi implants does not require the mixing of chemicals; uptake is passive, less damage to internal stem tissues; and less risk from phytotoxicity due to slow release of active chemicals Phosphite implants make phosphite treatment more practical and available within the wider community. Acquiring ground data & airborne imagery concurrently CIR image of study area showing 23 sites Range in crown condition as measured using the indices Crown Density (CD), Foliage Transparency (FT), Crown Dieback Ratio (CDR) and Epicormic Index (EI) and the composite Total Crown Health Index (THCI). (A) CD = 15%, FT=65%, CDR=60%, EI=%, TCHI=23% (B) CD=30, FT=35%, CDR=55%, EI=95%, TCHI=36% (C) CD=60%, FT=35%, CDR=15%, EI=30%, THCI=70% (D) CD=80%, FT=15%, CDR=0%, EI=0%, THCI=91%. Mean Total Crown Health Index (TCHI) ± SE for all sites captured with the Digital Multispectral Imagery showing TCHI varied between 28±7 (Site 19) and 86±1 (Site 13). 8

9 Delineation of tree crowns & extraction of data Figure LHS. CIR image of the DMSI showing the manually delineated crowns of unhealthy trees within site 19 (A) and healthy trees within site 13 (B). The crowns within the healthy site have a brighter and more uniform appearance when compared to the unhealthy site. Remote sensing of declines in tree health This site features a large amount of dead wood (brown). This true colour image doesn t tell us much about our trees though! Spectral signatures of trees within site 19 (unhealthy) and site 13 (healthy) showing differences at Band 2 (Green 550nm) and Band 4 (NIR 780nm). Site 13 Site 19 Eucalyptus gomphocephala tuart woodland Severe Decline: Site 8 More than half the large tuarts in this image are showing signs of decline Yalgorup National Park Change Composite Image Between 2008 and 2010 DMSI 0.5 m pixels Courtesy of SpecTerra Services Pty Ltd Change in REEI: Site Crown Mean REEI Change over ? The bright aqua colour reflects the growth of these trees over the period. Generally, the site appears to be suffering with patches of red dominating the bottom of the image Why is this so? These trees were injected with minerals Selecting for resistance Eucalyptus marginata story We know that resistance of jarrah to Pc is under strong genetic control. Clones produced from plants: 1. Mature trees on sites infested with ihp. c 2. Mature trees showing resistance to leafminer 3. Seedlings surviving inoculation with Pc in pots Half sib families of 14 month old pot grown jarrah stem inoculated Necrotic lesions measured Stems pruned to remove diseased tissue Healthy axillary growth used for tissue culture Relative levels of resistance or susceptibility of 16 seedling families determined from lesion lengths Families with mean lesion lengths significantly smaller that a standard susceptible family were designated as resistant Within families individual seedlings showing outstanding resistance (restricted lesions) or high susceptibility (long lesions) were selected. Those selected for cloning were: RR: resistant individuals from resistant families RS: apparently resistant individuals from susceptible families SS: Susceptible individuals for susceptible families 9

10 (Stukely et al 2007 Forest Ecol & Management 238; ) (a) Mortality of clones of Eucalyptus marginata, in the three resistance categories RR, RS, and SS, at 1, 2, 7, and 13 years after they were transplanted to the field and inoculated with Phytophthora cinnamomi. The clones are ranked on their mortality after 13 years. The resistance category relates to the selected inoculated seedling from which the clone was propagated: resistant individuals from resistant families (RR), apparently resistant individuals from susceptible families (RS), and susceptible individuals from susceptible families (SS). (Stukely et al 2007 Forest Ecol & Management 238; ) GENOME PORTAL How does Phosphite induce plant defence? Stuart Pearse Ricarda Jost Plant physiology Mol. plant biology physiological effects of Phi Phi detection + metabolomics Screen for P i sensitivity / Pc susceptibility Hakea / Grevillea spp. Genetics of P i sensitivity / Pc susceptibility Marker development Hybrids insensitive to P i and resistant to Pc physiology proteomics metabolomics genomics genetic screening physiological effects of Phi Phi effects on P i signalling networks Phi induced proteome changes Arabidopsis acc. Phi dept. metabolite changes targets for Phi action nat. diversity in P i sensitivity / Pc susceptibility psf.org/phyci1/phyci1.home.html Plant genetics Xuanli Ma Mol. plant biology Oliver Berkowitz THANK YOU Lupinus albus (White lupins) biomass (grams) at different phosphite application rates (kg/ha): control ( ), 120 ( ), 240 ( ), 480 ( ), 720( ), and, 1440 ( ). Bars represent the standard error of the means. Each treatment had N=9 pots with 3 seeds sown in each pot. All the mature plants were harvested 35 days after sowing. ass (g) Mean ma Roots Stems Leaves Total Mass 10

11 P. humicola P. inundata P. aff. humicola P. taxon personii P. gemini P. taxon cyperaceae P. rosacearum P. rosacearum Evidence of FISH specificity. Probe binding to the nuclei of Phytophthora cinnamomi (yellow arrows as examples) resulting in a spotted fluorescence. The oospores likely to be a Pythium species (white arrows) are dull or uniform in colour with associated nearby hyphae lacking probe binding (red arrows). P. aff. rosacearum P. taxon kwongan P. taxon cooljarloo P. taxon walnut Corresponding bright view of picture 2. The red arrows points to the hyphae of Pythium which were not visible in picture 2 due to the Phytophthora cinnamomi specific probe not binding to non P. cinnamomi structures. P. cinnamomi yellow arrows. Pythium white arrows. Response to injection with iron Tuart Novel and innovative technologies Before injection Six weeks After Injection HEALTHY FORESTS SYMPOSIUM 2011 Phytophthora species in natural ecosystems in WA New records for WA P. inundata P. taxon PgChlymado - clade 6 P. taxon asparagi - clade 6 P. taxon niederhauseria clade 7 P. taxon personii- clade 6 Numerous N new Phytophthora hth species described d P. multivora - clade 2 P. elongata - clade 2 P. arenaria - clade 4 P. constricta clade 9 P. thermophila - clade 6 P. gregata - clade 6 P. gibbosa - clade 6 P. fluvialis - clade 6 P. amnicola clade 6 P. bilorbang - clade 6 Lateral Flow Device for Phytophthora identification + ve for Phytophthora 11

12 Eradication Treatment strategy PCD Change Image , 1m resolution Using DMSI for change detection PCD ratio of infrared to red bands Change in PCD over 12 months Very powerful tool for spatial and temporal analysis of crown health May 2006, True Colour Image, 1m resolution (Stukely et al 2007 Forest Ecol & Management 238; ) The Scientific Questions the Genome Sequence will Help Address Investigate life cycle mechanisms of infection sporulation spore dormancy survival strategies virulence and host range Study the mechanism(s) of action of chemical control agents including phosphite Develop genomics tools for P. cinnamomi research. cpsm.murdoch.edu.au 12