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 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 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

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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. , Phytophthora multivora P. frigida B. attenuata, B. littoralis, B. menziesii, B. prionotes, P. bicheria Conospermum sp., Leucopogon verticillatus, P. elongata 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, , Andersonia sp., Patersonia xanthia, grandis, Xanthorrhoes preissii, X. gracilis, Leucopogon propinquus Dryandra squarrosa Only on lateritic soils in WA and in Victoria on sandy soils

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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 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?

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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 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

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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 ddiespite bbieing 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

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Germination of thick walled chlamydospore with multiple germ tubes. 3 4

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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

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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 •Many annual and herbaceous perennials are symptomless • P. cinnamomi may be present in an area with no hosts of P. cinnamomi susceptible woody species, surviving in annual and • Haustoria indicating biotrophic mode herbaceous perennials

•Ability to grow as a biotroph without ‘moving’ into a necrotrophic • It is possible to eradicate P. cinnamomi from black mode. gravel sites by removing all living vegetation including annuals and hbherbaceous perennials • Potential endophytic mode in some hosts •Eradication may take at least 2.5‐3 years, so all • Numerous selfed oospores produced in some hosts dormant structures have germinated and died in •Stromata common: used as a nutrient ‘springboard’ for oospore and absence of host plants. chlamydospore production. • After this time revegetation may be undertaken in •Extremely ‘plastic and versatile’ pathogen 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 total P as affected by Phi accumulation ‐ there 3‐ was no significant difference upper (■)and lower Soil plant‐available P (PO4 ) as affected by Phi accumulation. Upper horizon (■)was significantly different from lower horizon (■). horizons (■).

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Phosphite treated P. c –free plots Phosphite treated Banksia attenuata mature (■) and senesced (■)leaves P. c plots had significantly higher total P levels

0.16 0.14 Non‐phosphite 0.12 P. c‐free plots 0.1 0.08

phosphorus (mg/g) phosphorus 0.06

0.04 Non‐phosphite 0.02 P. c plots Mean total 0 Non-phosphite sprayed and P. cinnamomi- Phosphite sprayed and P. cinnamomi-free free Treatments

Similar trends seen for Adenanthos cuneatus, Banksia coccinea and Jacksonia spinosa Species diversity as affected by PC and Phi interaction in GRNP (nMDS plot)

. Phosphite trial – liquid injection vs powder capsule implants 70 60 50 40 30 20 10

Mean canopy Mean canopy closure (%) 0 PHI-PC- PHI-PC+ PHI+PC+ PHI+PC- Treatments

Control Mixed Phosphate Phosphite Phosphite Control Mixed Phosphate Phosphite Phosphite nutrient + nutrients implants liquid nutrient + nutrients implants liquid

• Results show that both phosphite liquid and implants are effective at controlling lesion extension in B. grandis and E. marginata, caused by P. Mean (%) canopy cover and accompanying hemispherical photographs of the cinnamomi. 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. • Phosphate and mixed nutrient treatments may help control Phytophthora infections by improving plant health.

Phosphite implants Acquiring ground data & airborne imagery concurrently

Composition per 1.0 g capsule Range in crown condition as measured using the indices Crown Density (CD), Foliage Transparency Capsule Constituents Composition % by weight Dose (mg/10 cm trunk circumference) (FT), Crown Dieback Ratio (CDR) and Epicormic Phosphite 58 580 Index (EI) and the composite Total Crown Health Phosphate 4 40 Index (THCI). Soluble potash 38 380 (A) CD = 15%, FT=65%, CDR=60%, EI=100%, TCHI=23% (B) CD=30, FT=35%, CDR=55%, EI=95%, TCHI=36% Phosphite application – liquid/soluble implant pros and (C) CD=60%, FT=35%, CDR=15%, EI=30%, THCI=70% cons (()D) CD=80%, FT=15%, CDR=0%, EI=0%, THCI=91%.

Compared to phosphite liquid, phosphite implants: Control • 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 Mean Total Crown Health Index (TCHI) ± SE forallsitescapturedwiththeDigital Phosphite implants make phosphite treatment more practical and available Multispectral Imagery showing TCHI varied CIR image of study area showing 23 sites within the wider community. between 28±7 (Site 19) and 86±1 (Site 13).

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Delineation of tree crowns & extraction of data Remote sensing of declines in tree health Figure LHS. CIR image of the DMSI showing the manually delineated crowns of unhealthy trees within site 19 (A) and healthy trees within site This site features a 13 (B). The crowns within the healthy site have a large amount of brighter and more uniform appearance when dead wood compared to the unhealthy site. (brown).

This true colour image doesn’t tell us much about our trees though!

Spectral signatures of trees within site Site 13 19 (unhealthy) and site 13 (healthy) showing differences at Band 2 (Green 550nm) and Band 4 (NIR 780nm).

Site 19 Eucalyptus gomphocephala ‐ tuart woodland

Severe Decline: Site 8 Change in REEI: Site 3 3 Crown Mean REEI 304 2.5 More than half the 305 2 301 large tuarts in this 302 1.5 image are showing 303 804 303 signs of decline. 1 304 305 0.5 801 803 302 0 2007 2008 2009 2010 301 Change over 2008‐2010? 802 • The bright aqua colour reflects the Yalgorup National Park growth of these trees over the 2008 – 2010 Change Composite period. Image • Generally, the site appears to be suffering Between 2008 and 2010 with patches of red dominating the bottom DMSI 0.5 m pixels of the image

Courtesy of SpecTerra Services Why is this so? Pty Ltd These trees were injected with minerals.

Selecting for resistance‐ • Half‐sib families of 14 month‐old pot grown jarrah stem inoculated story • Necrotic lesions measured • Stems pruned to remove diseased tissue We know that resistance of jarrah to Pc is under • Healthy axillary growth used for tissue culture strong genetic control. • Relative levels of ‘resistance’ or susceptibility of 16 seedling families determined from lesion lengths Clones produced from plants: • Families with mean lesion lengths significantly smaller that a standard susceptible family were designated as ‘resistant’ 1. Mature trees on sites ifinfested wiihth P. c • Within families individual seedlings showing outstanding resistance (restricted lesions) or high susceptibility (long lesions) were 2. Mature trees showing resistance to leafminer selected. 3. Seedlings surviving inoculation with Pc in Those selected for cloning were: RR: resistant individuals from resistant families pots RS: apparently resistant individuals from susceptible families SS: Susceptible individuals for susceptible families

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(Stuk el y et al 2007 Forest Ecol & Management 238; 330‐334)

(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; 330‐334)

How does Phosphite induce plant defence? GENOME PORTAL Stuart Pearse Ricarda Jost Plant physiology Mol. plant biology

physiological effects of Phi physiological effects of Phi physiology

Phi detection + metabolomics Phi effects on Pi signalling networks

Screen for Pi sensitivity / Pc susceptibility proteomics Phi induced proteome changes

Hakea / Grevillea spp. metabolomics Arabidopsis acc.

Genetics of Pi sensitivity / Pc susceptibility genomics Phi dept. metabolite changes Marker development targets for Phi action genetic screening Hybrids insensitive to Pi and resistant to Pc nat. diversity in Pi sensitivity / Pc susceptibility

Plant genetics Mol. plant biology http://genome.jgi‐psf.org/Phyci1/Phyci1.home.html Xuanli Ma Oliver Berkowitz

Lupinus albus (White lupins) biomass (grams) at different THANK YOU 1.20 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 1.00 each pot. All the mature plants were harvested 35 days after sowing.

0.80 ss (g) a 0600.60 Mean m

0.40

0.20

0.00 Roots Stems Leaves Total Mass

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P. humicola P. inundata P. aff. humicola P. taxon personii 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. gemini P. taxon cyperaceae P. rosacearum P. rosacearum

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 P. aff. rosacearum P. taxon kwongan P. taxon cooljarloo P. taxon walnut 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 Lateral Flow Device for Phytophthora WA identification •New records for WA P. inundata P. taxon PgChlymado - clade 6 + ve for Phytophthora P. taxon asparagi - clade 6 P. taxon niederhauseria clade 7 P. taxon personii- clade 6 •Numerous new Phyt ophth ora species descr ibdibed 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

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Eradication ‐ Treatment strategy Using DMSI for change detection PCD Change Image 2006-2005, 1m resolution 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

The Scientific Questions the Genome Sequence will Help Address • Investigate – life cycle – mechanisms of infection – sporulation – spore dormancy (kl(Stukely et al 2007 – survival strategies Forest Ecol & – virulence and host range Management • Study the mechanism(s) of action of chemical control 238; 330‐334) agents including phosphite • Develop genomics tools for P. cinnamomi research.

cpsm.murdoch.edu.au

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