Response of selected South Australian native plant species to Phytophthora cinnamomi

Authors

  • K. H. Kueh,

    1. Waite Research Institute, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA5064
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    • Present address: Agriculture Research Centre, 93720 Kuching, Sarawak, Malaysia.

  • S. F. McKay,

    1. Waite Research Institute, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA5064
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  • E. Facelli,

    1. Waite Research Institute, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA5064
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  • J. M. Facelli,

    1. School of Earth and Environmental Sciences, The University of Adelaide, Adelaide, SA5005
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  • R. M. A. Velzeboer,

    1. Department of Environment and Natural Resources, PO Box 721, Victor Harbor, SA5211 Australia
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  • A. J. Able,

    1. Waite Research Institute, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA5064
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  • E. S. Scott

    Corresponding author
    1. Waite Research Institute, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA5064
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E-mail: eileen.scott@adelaide.edu.au

Abstract

Thirty-seven South Australian native plant species from 11 families, including 15 threatened species in the state (of which six are listed as threatened under the federal Environment Protection and Biodiversity Conservation Act 1999) were assessed for response to infection by Phytophthora cinnamomi. Seedlings, 3–6 months old and grown in a greenhouse, were inoculated by placing infested pine wood plugs in the potting mix, maintained in moist conditions and assessed for mortality and disease symptoms for between 3 and 10 months. Thirty species were found to be susceptible, of which nine were highly susceptible, 15 moderately susceptible and six slightly susceptible. Three species were found to be resistant and results for four species were inconclusive. Six of the 15 threatened, rare or locally endangered species tested (Eucalyptus viminalis var. viminalis, Correa aemula, C. calycina, Olearia pannosa ssp. pannosa, Pomaderris halmaturina ssp. halmaturina and Prostanthera eurybioides) were moderately susceptible, while two (Allocasuarina robusta and Pultenaea graveolens) were highly susceptible. Significant populations of at least five of the threatened species susceptible to the disease are located close to confirmed or suspected Phytophthora-infested areas or growing in areas conducive for P. cinnamomi. An effective management strategy is therefore required to avoid extinction of such species due to infection by the phytophthora dieback pathogen.

Introduction

Phytophthora dieback is a soilborne disease which causes damage to many horticultural crops worldwide (Erwin & Ribeiro, 1996). In Australia, the disease also causes damage of epidemic proportions to native vegetation in many regions, particularly in the south-eastern and south-western regions of the country. The disease was first associated with the death of a large number of native plants in the jarrah forests in Western Australia in 1921 (Podger, 1972) and in 1952 in Gippsland in the south-east of Victoria (Marks et al., 1972). By the 1990s, the disease had destroyed large areas of native vegetation in Western Australia, Tasmania, Queensland and Victoria, causing local susceptible species to disappear (Weste, 1986). Phytophthora dieback continues to threaten the survival of susceptible native plant species, particularly endangered species such as Banksia brownii in Western Australia and Wollemia in New South Wales (O’Gara et al., 2005; Reiter et al., 2004). As such, phytophthora dieback is recognized as a key threatening process to the natural ecosystem under the Environment Protection and Biodiversity Conservation (EPBC) Act 1999 of Australia.

The disease is caused mainly by Phytophthora cinnamomi, which causes root and crown rots, resulting in shoot dieback, chlorotic foliage and defoliation. When rot has girdled the collar region, plants invariably wilt and die. Young eucalypt seedlings tend to show greater sensitivity to phytophthora dieback than older plants (Peace, 1962). The spread and severity of disease depends very much on the prevailing weather conditions (Tregonning & Fagg, 1984). Being a water mould, P. cinnamomi thrives in warm and wet conditions, when the pathogen sporulates and releases motile zoospores which are attracted to and infect the roots. Infected plants may die during hot, dry spells following wet weather, as plants with rotted roots are unable to take up sufficient water to compensate for increased water loss. This pattern is common in the Mediterranean-type environments of the south-western and south-eastern parts of Australia where a warm and wet spring is followed by a hot and dry summer.

Phytophthora cinnamomi affects a large number of species in Australia’s diverse and unique flora, from large trees to prostrate shrubs (Barker & Wardlaw, 1995; Shearer et al., 2004). Barker & Wardlaw (1995) found that 36 of 47 species native to Tasmania tested were susceptible. Assessment of the susceptibility of 749 native plant species from 253 genera in Western Australia led Shearer et al. (2004) to estimate 40% of the 5710 described plant species in the South-West Botanical Province to be susceptible to this pathogen. Of these, about 800 species were ranked as highly susceptible. A field evaluation of susceptibility by Weste (1986) showed that up to 75% of the native flora in an infested native vegetation site in Brisbane Ranges National Park, Victoria, was susceptible. The disease caused not only local extinction of susceptible species and endangered the continued survival of rare species but also changed the species composition of the plant communities (Weste, 1986).

In comparison, very little is known about the susceptibility of the native flora of South Australia to this pathogen. Phytophthora cinnamomi was first isolated in South Australia in 1969 (Davison, 1970) and, since then, has been found extensively in many areas of native vegetation with high conservation value, such as the Mount Lofty Ranges, Fleurieu Peninsula (Lee & Wicks, 1977) and Kangaroo Island (Williams et al., 2007). While P. citricola, P. cryptogea, P. drechsleri and P. megasperma have been isolated from forest nurseries and pine plantations (Davison & Bumbieris, 1973; Pratt & Heather, 1973), and have the potential to cause dieback of native plants such as Banksia and Eucalyptus spp. (Weste, 1975; Shearer et al., 1987), P. cinnamomi is considered to cause most damage to native vegetation in South Australia (Velzeboer et al., 2005).

Protection of native flora from phytophthora diseases has relied on hygiene measures, such as provision of car wash-down facilities and restriction of access to conservation areas during wet periods, to prevent or reduce the spread of the pathogen to non-infested areas. However, in areas where the pathogen has already spread widely, hygiene measures alone are no longer effective to protect the vegetation from the disease. More effective and targeted strategies require evaluation of the susceptibility of native plant species to phytophthora dieback and prioritization for protection accordingly. Velzeboer et al. (2005) reported the potential risk posed by phytophthora dieback to 145 threatened South Australian native plant species according to their proximity to an infestation and vulnerability to infection by species of Phytophthora. However, reliable information about the effect of P. cinnamomi on South Australian native plant species, and threatened plant species in particular, is very limited. Most of the species in South Australia assumed to be susceptible have been designated as such based on observation of disease symptoms in the field or isolation of P. cinnamomi from root and soil samples. In many cases, P. cinnamomi was not confirmed as the cause of disease. This lack of knowledge has hampered the management of phytophthora dieback in South Australia (Velzeboer et al., 2005). Therefore, the aim of this study was to assess the susceptibility of selected South Australian native plant species to P. cinnamomi in a greenhouse, where conditions were conducive for disease.

Materials and methods

Experiments were carried out to assess the susceptibility of South Australian native plant species to P. cinnamomi and to assess the effect of seedling age on susceptibility to infection by P. cinnamomi. The method of inoculation was adapted from Shearer et al. (2004). Based on the results of preliminary experiments, seedlings were planted in limed University of California (UC) mix, unless otherwise stated, and the soil inoculated with infested pine wood plugs.

Testing the susceptibility of South Australian native plant species

Preparation of planting material

Plant species native to South Australia were selected for testing on the basis of a list of threatened species compiled by Velzeboer et al. (2005), their relative importance in the communities of the Mount Lofty Ranges and availability of seeds. Seeds and seedlings for the experiments were obtained from the Seed Conservation Centre, Adelaide Botanic Gardens and Blackwood Nursery, South Australia. Although seeds of 45 species were sown in potting mix, only 37 species, including 15 threatened species, were successfully raised in numbers sufficient for testing. Two species, Prostanthera eurybioides and Correa decumbens were prepared through cuttings. Cuttings of Pr. eurybioides were obtained from five plants raised from seeds obtained from the Seed Conservation Centre, while C. decumbens was propagated through cuttings obtained from a small population at Mount Bold Conservation Park (35°05′ 06·11″ S, 138°43′ 10·83″ E, 270 m a.s.l.). As the numbers of seedlings were limited and obtained at different times, susceptibility testing was carried out in batches and with different numbers of replicates. In all, 37 species from 11 families were assessed, comprising 22 common, six endangered, four vulnerable, four rare and one uncommon species (Velzeboer et al., 2005) (Table 1). Seeds of Eucalyptus sieberi, a known susceptible species, were purchased from Forest Products Commission, Western Australia and included as a susceptible control, where disease was expected to develop after inoculation.

Table 1.   List of South Australian native plant species, arranged by conservation status, tested for susceptibility to Phytophthora cinnamomi
No.SpeciesCommon nameConservation statusaFamily
EPBC Act 1999NPW Act 1972Other
  1. aSpecies listed as rare (R), vulnerable (V) or endangered (E) under the National Parks and Wildlife (NPW) Act 1972, South Australia (Velzeboer et al., 2005) and Environment Protection and Biodiversity Conservation (EPBC) Act 1999.

  2. bSeeds or seedlings of the species obtained from Seed Conservation Centre, Adelaide.

  3. cSpecies has no listing under legislation but is of conservation concern in the Mount Lofty Ranges; listed as uncommon (U) by Ainsley & Guerin (2009).

  4. dSeeds obtained from Blackwood Nursery, South Australia.

Threatened species
 1 Acacia enterocarpa b Jumping-jack wattleEE Fabaceae
 2 Acacia pinguifolia b Fat-leaf wattleEE Fabaceae
 3 Allocasuarina robusta b Mount Compass oak-bush E Casuarinaceae
 4 Brachyscome diversifolia b Tall daisy E Asteraceae
 5 Oreomyrrhis eriopoda b Australian carraway E Umbelliferae
 6 Prostanthera eurybioides b Monarto mintbushEE Lamiaceae
 7 Correa calycina var. calycinabHindmarsh correaVV Rutaceae
 8 Glycine tabacina b Variable glycine V Fabaceae
 9 Olearia pannosa ssp. pannosabSilver daisy-bushVV Compositae
10 Pomaderris halmaturina ssp. halmaturinabKangaroo Island pomaderrisVV Rhamnaceae
11 Acacia spooneri b Nectar brook wattle R Fabaceae
12 Correa aemula b Hairy correa R Rutaceae
13 Eucalyptus dalrympleana ssp. dalrympleanabMountain gum R Myrtaceae
14 Eucalyptus viminalis var. viminalisbManna gum R Myrtaceae
15 Pultenaea graveolens b,c Scented bushpea  UFabaceae
Common species
16 Acacia leiophylla b Golden coastal wattle   Fabaceae
17 Acacia melanoxylon b Black wattle   Fabaceae
18 Acacia paradoxa b Prickly wattle   Fabaceae
19 Acacia verniciflua b Varnish wattle   Fabaceae
20 Allocasuarina verticillata d Drooping sheoak   Casuarinaceae
21 Allocasuarina muelleriana d Slaty sheoak   Casuarinaceae
22 Austrodanthonia carphoides d Wallaby grass   Gramineae
23 Banksia marginata d Silver banksia   Proteaceae
24 Correa decumbens Spreading correa   Rutaceae
25 Hakea rostrata d Beaked hakea   Proteaceae
26 Hakea rugosa d Dwarf hakea   Proteaceae
27 Kunzea pomifera b Muntries   Myrtaceae
28 Eucalyptus baxteri d Brown stringybark   Myrtaceae
29 Eucalyptus cladocalyx b Sugar gum   Myrtaceae
30 Eucalyptus microcarpa b Grey box   Myrtaceae
31 Eucalyptus odorata b Peppermint box   Myrtaceae
32 Leptospermum continentale d Prickly tea tree   Myrtaceae
33 Leptospermum juniperinum d Prickly tea tree   Myrtaceae
34 Leptospermum myrsinoides d Heath tea-tree   Myrtaceae
35 Platylobium obtusangulum d Common flatpea   Fabaceae
36 Pultenaea daphnoides b Large-leaf bush pea   Fabaceae
37 Pultenaea largiflorens b Twiggy bush-pea   Fabaceae

All experimental plants were maintained in a greenhouse at the Waite Campus, South Australia (34°58′ 15·13″ S, 138°38′ 25·18″ E, 140 m a.s.l.). Seedlings were raised in potting mix for native species and later transplanted to 15-cm diameter, free-draining pots filled with limed UC mix with a pH of about 6·5. UC mix was prepared as described by Naseri et al. (2008). At the time of transplanting, 3 g of Osmocote (Scotts Australia Pty Ltd), a slow release fertilizer for Australian native plants with formulation 17 N:1·6 P:8·7 K + trace elements, were applied to each pot. No fertilizer was applied thereafter. Six species (Pr. eurybioides, Pultenaea graveolens, Pomaderris halmaturina ssp. halmaturina, Olearia pannosa ssp. pannosa, Oreomyrrhis eriopoda and Glycine tabacina) failed to thrive in UC mix. Freshly prepared seedlings or cuttings were transplanted into BioGro® native potting mix (BioGro). Allocasuarina robusta was tested twice in UC mix at different times and Pr. eurybioides was tested twice in UC mix and once in BioGro® to determine if soil type was likely to have a major effect on response to inoculation with P. cinnamomi.

At transplanting, two centrifuge tubes (1 × 10 cm) were inserted close to the roots on opposite sides of the plant to allow for subsequent placement of inoculum without disturbing the substrate or the roots. The pots were watered daily to saturation and left to drain. Temperature in the greenhouse fluctuated between 17 and 29°C depending on the season. During summer when sunlight was strong, shade cloth was used to reduce sunlight by 50%. The plants were maintained in this way until inoculation.

Phytophthora cinnamomi isolates

Phytophthora cinnamomi isolates 71a and SC4, both from South Australia, were used for susceptibility testing. Isolate 71a was obtained from a soil sample collected from around the roots of a dead Xanthorrhoea semiplana ssp. tateana on Kangaroo Island (35°47′ 55·78″ S, 137°27′ 33·27″ E, 62 m a.s.l.) on 5 October 2007 (Williams et al., 2007) and SC4 was isolated from soil collected around a dead X. semiplana in Scott Creek Conservation Park (35°05′ 10·24″ S, 138°41′ 52·98″ E, 380 m a.s.l.) on 3 June 2008. Both isolates were confirmed as P. cinnamomi in June 2008 using a polymerase chain reaction assay at the Centre for Phytophthora Science and Management, Murdoch University, Western Australia, and diagnostic sequences of the internal transcribed spacer regions submitted to GenBank (accession numbers JQ306322 and JQ306323).

Preparation of infested pine wood plugs

Live branches, about 1·0–1·5 cm in diameter, were obtained from mature Pinus radiata trees, processed and inoculated with P. cinnamomi as described by Butcher et al. (1984). Infested pine plugs for isolates 71a and SC4 were prepared separately.

Inoculation of pots

When seedlings were 3–6 months old, the centrifuge tubes were removed from the pots and two pine plugs infested with P. cinnamomi isolate 71a were inserted into one hole and two infested with isolate SC4 were inserted into the other hole. Between four and 15 plants of each species were inoculated. Eucalyptus sieberi was included as a susceptible control. Up to five plants of each species, including E. sieberi, were mock-inoculated with sterilized blank pine plugs as pathogen-free controls. After inoculation, the pots containing the plants were placed in 2-L plastic containers and flooded with tap water for 48 h to stimulate production of zoospores. The water was then decanted such that up to 4 cm depth was maintained in the container to keep the potting mix moist. A disinfested probe (G Bug, Measurement Engineering Australia) was later inserted into one pot to measure the water potential of the potting mix for the duration of the experiment. Water potential was always in the range of −1 to −9 kPa.

Plants were arranged in a randomized complete block design and watered daily. All plants were observed daily for dieback symptoms and date of plant death was recorded. Altogether, the 37 plant species were tested in seven batches and all plants tested were inoculated in winter and spring 2010, except for batch 3 which was inoculated in summer (January).

Effect of seedling age on susceptibility to phytophthora dieback

Six species that differed in susceptibility, as determined in this study, were selected to examine the effect of seedling age on the response to infection by P. cinnamomi. Seeds of Eucalyptus goniocalyx, Eucalyptus viminalis var. viminalis, Eucalyptus cladocalyx, Al. robusta, Kunzea pomifera and Acacia paradoxa were procured from Blackwood Nursery (South Australia), sown in potting mix in March and June 2010 and seedlings maintained as described above. Seedlings 100 and 180 days from the date of sowing were inoculated with infested pine plugs on 6 October 2010, 15 replicates of each species. Another five plants of each age and species were inoculated with blank plugs as pathogen-free controls. Eucalyptus sieberi seedlings, 70 days old, were inoculated as susceptible controls. All plants were arranged in a randomized complete block and monitored for disease symptoms and mortality for 12 weeks.

Data collection

Plants (whether inoculated or control) were harvested at death and the date recorded. At the time of harvest, soil was baited to assess presence of P. cinnamomi. About 30 g of the potting mix together with root pieces from each pot were collected in a plastic cup containing 150 mL of reverse osmosis (RO) water. Five to six pairs of cotyledons of 3-week-old E. sieberi were floated on the resulting soil suspension as bait for P. cinnamomi (Marks & Kassaby, 1974). Phytophthora cinnamomi was assumed to be present if diagnostic sporangia could be seen growing from the edges of cotyledons under ×100 magnification after incubation in the dark at ambient temperature for 4–8 days. The harvested plants were washed under running tap water and the roots were examined for lesions and rot. Sections of fine and lateral roots and the collar region with lesions were excised, surface sterilized in 1% sodium hypochlorite for 1 min, rinsed three times in sterile distilled water and allowed to dry on a sterile paper towel. If no lesion was observed, root sections were selected at random. The epidermis of the roots was removed and both ends of each section, which might have absorbed sodium hypochlorite, were cut and discarded. The root sections were cut in half and the cut surface gently pressed onto Phytophthora-selective medium, P10ARPH (O’Gara et al., 1996), to ensure good contact with the medium. The collar region was prepared in a similar manner. Generally about 10–12 pieces of root tissue from each plant were plated. If coenocytic hyphae with numerous coralloid structures and spherical swellings could be observed to emerge from the root tissue within 1–2 days of incubation in the dark at 25°C, P. cinnamomi was assumed to be present. If tissue samples, particularly from the collar region, collected at the time of plant death yielded P. cinnamomi, then phytophthora root rot was considered to be the cause of death; otherwise, the plant was considered as censored in the calculation of mortality (see below). Reisolation of P. cinnamomi from root tissue was considered part of the definition of mortality, to reduce the risk of misclassifying a response as susceptible when the cause of death was, for example, excessively moist soil (Davison & Tay, 1987).

At the end of each experiment, all surviving plants were harvested (and treated as censored in the analyses), washed and roots examined for symptoms, such as lesions. Root samples were collected from each plant to test for the presence of P. cinnamomi and all potting mix from each pot was baited as described above.

Statistical analysis

Percentage plant mortality due to phytophthora dieback was calculated based on the number of dead plants from which P. cinnamomi was reisolated from root samples and the total number of plants inoculated. Kaplan–Meier survival curves of the inoculated and the non-inoculated plants within the same species were compared using a log-rank (Mantel–Cox) test performed using graphpad prism (version 5.04 for Windows; GraphPad Software) at  0·05. Kaplan–Meier survival curves were prepared for all species in batches 1, 2 and 3, except where control plants were not included, viz. E. odorata and C. aemula, for which only four plants were available. Percentage plant mortality and severity of disease symptoms observed were used to categorize species as highly susceptible, moderately susceptible, slightly susceptible, resistant but host and resistant and non-host, as described by Barker & Wardlaw (1995).

Results

Disease symptoms on positive control, E. sieberi

The high mortality in the positive control indicated that the P. cinnamomi isolates used were pathogenic (Table 2). Inoculated plants of E. sieberi started to wilt and die 2 weeks after inoculation and mortality increased steeply in the following 6 weeks (Figs 1–3). By the 17th week after inoculation, over 80% of the inoculated E. sieberi had died in all three batches. Plants wilted suddenly, followed by rapid death. Roots of all dead plants had rotted completely and turned dark brown. Neither leaf chlorosis nor defoliation was observed prior to plant death. Phytophthora cinnamomi was readily and consistently reisolated from this material (Table 2). Soil samples, collected when each plant died, consistently showed the presence of P. cinnamomi (Table 3). One non-inoculated E. sieberi plant in batch 1 and two in batch 2 died but P. cinnamomi was not reisolated from root samples. Comparison of the survival curves showed that mortality of inoculated E. sieberi seedlings was significantly greater (< 0·01) than that of non-inoculated seedlings (Table 2). The survival curves of inoculated E. sieberi in the three batches were similar (> 0·05). Between 71·4 and 100% of all potting mix samples from pots that contained inoculated E. sieberi yielded P. cinnamomi (Table 3). The pathogen was not isolated from the non-inoculated controls.

Table 2.   Susceptibility of selected South Australian native plants to Phytophthora cinnamomi
No.SpeciesaMortality (%)bNo. inoculatedNo. deadNo. dead plants yielding P. cinnamomiNo. survivingplants yielding P. cinnamomiAverage survival time and range (weeks)Log-rank (Mantel–Cox) testcNo. control plantsNo. control plants deaddCategory of susceptibilitye
  1. aThe 37 plant species were tested in batches and Eucalyptus sieberi was included within batches 1–3 as a susceptible control. Plants were grown in UC potting mix unless shown otherwise.

  2. bMortality (%) calculated based on number of plants dead after first inoculation and which yielded P. cinnamomi.

  3. cLog-rank (Mantel–Cox) was used to test difference between the survival curves of the inoculated and non-inoculated plants within the same species at  0·05. (S, significant difference; NS, no significant difference).

  4. d P. cinnamomi was not isolated from roots or potting mix from any of the non-inoculated controls.

  5. eHS: highly susceptible; mortality due to P. cinnamomi was ≥80%; disease symptoms included sudden wilt, extensive root and collar rot, rapid death; P. cinnamomi reisolated from root tissue. MS: moderately susceptible; mortality due to P. cinnamomi ranged from 20 to <80%; disease symptoms included slow dieback, variable root and collar rot; P. cinnamomi reisolated from root tissue. SS: slightly susceptible; mortality due to P. cinnamomi was <20%; disease symptoms included localized lesions on fine roots; P. cinnamomi reisolated from root tissue. RH: resistant but host; mortality due to P. cinnamomi was zero; inoculated plants remained healthy but P. cinnamomi reisolated from root tissue. R: resistant and non-host; mortality due to P. cinnamomi was zero; inoculated plants remained healthy and P. cinnamomi was not reisolated from root tissue. IC: inconclusive result due to death of both inoculated and non-inoculated plants.

  6. fPlants were grown in BioGro® potting mix for Australian native plants (BioGro).

Batch 1 (inoculated in spring)
 1 Leptospermum continentale 26·715441016·0 (7–21)NS50MS
 2 Leptospermum juniperinum 33·31555211·4 (7–21)NS50MS
 3 Acacia paradoxa 6·71511125 (5–21)NS50SS
 4 Acacia melanoxylon 6·715111121NS50SS
 5 Acacia verniciflua 46·7158769·6 (4–15)NS50MS
 6 Correa decumbens 66·715131009·3 (5–14)S50MS
 7 Prostanthera eurybioides 46·71577614·1 (4–21)NS30MS
 8 Platylobium obtusangulum 83·366507 (3–16)NS22HS
  Eucalyptus sieberi 86·715151304·8 (2–5)S51 
Batch 2 (inoculated in spring)
 9 Eucalyptus microcarpa 14·3711618NS30SS
10 Eucalyptus dalrympleana 13·31532116 (5–18)NS51SS
11 Eucalyptus baxteri 86·715151305·3 (3–9)S31HS
12 Eucalyptus cladocalyx 20·015431010·5 (5–18)NS40MS
13 Eucalyptus viminalis var. viminalis20·01022716·5 (16–17)NS31MS
14 Eucalyptus odorata 25·04111500MS
15 Acacia leiophylla 13·31542512·8 (8–18)NS50SS
16 Leptospermum myrsinoides 26·71555914·4 (11–18)NS41MS
17 Pultenaea daphnoides 10055506·8 (2–10)NS31HS
18 Pultenaea largiflorens 80·055403S30HS
19 Pultenaea graveolens f 87·588706·3 (4–8)S41HS
20 Correa aemula 50·044202·7 (2–4)00MS
  Eucalyptus sieberi 80·0151412112·5 (8–17)S52 
Batch 3 (inoculated in summer)
21 Acacia enterocarpa 0400212NS30RH
22 Acacia pinguifolia 0500212NS30RH
23 Acacia spooneri 6·715111112NS50SS
24 Allocasuarina robusta 80·015141205·5 (3–12)S51HS
25 Brachyscome diversifolia 0920012NS30R
26 Banksia marginata 10015151506·3 (3–12)S52HS
27 Kunzea pomifera 93·315151406 (3–12)S52HS
  Eucalyptus sieberi 10015151506·4 (3–12)S50 
Batch 4 (inoculated in winter)
28 Allocasuarina robusta 201032711·7 (7–20)NS103MS
29 Austrodanthonia carphoides 010100031·0 (20–36)1010IC
Batch 5 (inoculated in winter)
30 Allocasuarina verticillata 33·31565529·7 (27–36)NS50MS
31 Allocasuarina muelleriana 10015151507 (4–12)S50HS
Batch 6 (inoculated in winter)
32 Prostanthera eurybioides 73·315141108·6 (3–29)NS52MS
33 Correa calycina 20·0157308·6 (3–29)NS52MS
34 Hakea rostrata 30·81374215·1 (3–44)NS54MS
35 Hakea rugosa 11·1961027·6 (3–44)S33IC
Batch 7 (inoculated in winter)
36 Pomaderris halmaturina ssp. halmaturinaf40·01064419·7 (4–29)NS51MS
37 Olearia pannosa ssp. pannosaf45·51185321·3 (3–41)NS31MS
38 Oreomyrrhis eriopoda f 10·010101024·4 (4–34)NS43IC
39 Prostanthera eurybioides f 46·715157025·3 (3–35)NS54MS
40 Glycine tabacina f 015150025·3 (3–35)NS43IC
Figure 1.

 Response of the eight plant species in batch 1 to infection by Phytophthora cinnamomi. The plants were inoculated by inserting pine wood plugs infested with P. cinnamomi into the potting mix on 3 October 2010 and assessed 21 weeks later. Each figure shows the Kaplan–Meier survival estimates for the inoculated and non-inoculated plants within the same species. The numbers of replicates of the inoculated and non-inoculated plants within each species and the significance of the log-rank (Mantel–Cox) test are given in Table 2. The species, in order of increasing susceptibility to phytophthora dieback, are: (a) Acacia melanoxylon; (b) Acacia paradoxa; (c) Leptospermum continentale; (d) Leptospermum juniperinum; (e) Prostanthera eurybioides; (f) Acacia verniciflua; (g) Correa decumbens; (h) Platylobium obtusangulum; (i) Eucalyptus sieberi as susceptible control. (___) control plants; (- - -) inoculated plants.

Figure 2.

 Response of the 10 plant species in batch 2 to infection by Phytophthora cinnamomi. The plants were inoculated by inserting pine wood plugs infested with P. cinnamomi into the potting mix on 16 November 2010 and assessed 18 weeks later. Each figure shows the Kaplan–Meier survival estimates for the inoculated and non-inoculated plants within the same species. The numbers of replicates of the inoculated and non-inoculated plants within each species and the significance of the log-rank (Mantel–Cox) test are given in Table 2. The species, in order of increasing susceptibility to phytophthora dieback, are (a) Acacia leiophylla; (b) Leptospermum myrsinoides; (c) Eucalyptus microcarpa; (d) Eucalyptus dalrympleana; (e) Eucalyptus cladocalyx; (f) Eucalyptus viminalis var. viminalis; (g) Eucalyptus baxteri; (h) Pultenaea largiflorens; (i) Pultenaea graveolens; (j) Pultenaea daphnoides; (k) Eucalyptus sieberi as susceptible control. (___) control plants; (- - -) inoculated plants.

Figure 3.

 Response of the seven plant species in batch 3 to infection by Phytophthora cinnamomi. The plants were inoculated by inserting pine wood plugs infested with P. cinnamomi into the potting mix on 6 January 2011 and assessed 12 weeks later. Each figure shows the Kaplan–Meier survival estimates for the inoculated and non-inoculated plants within the same species. The numbers of replicates of the inoculated and non-inoculated plants within each species and the significance of the log-rank (Mantel–Cox) test are given in Table 2. The species, in order of increasing susceptibility to phytophthora dieback, are (a) Acacia enterocarpa; (b) Acacia pinguifolia; (c) Acacia spooneri; (d) Brachyscome diversifolia; (e) Allocasuarina robusta; (f) Banksia marginata; (g) Kunzea pomifera; (h) Eucalyptus sieberi as susceptible control. (___) control plants: (- - -) inoculated plants.

Table 3.   Reisolation of Phytophthora cinnamomi from potting mix collected at the time of death of seedlings of South Australian native species and at end of experiments
SpeciesNumber of pots sampledNumber of samples yielding positive resultaSamples yielding positive result (%)
  1. aPotting mix suspension was baited with cotyledons of 3-week-old E. sieberi. If sporangia were produced, then P. cinnamomi was assumed to be present.

  2. P. cinnamomi was not isolated from roots or potting mix from any of the non-inoculated controls.

Batch 1
 Leptospermum continentale 151376·7
 Leptospermum juniperinum 15960·0
 Acacia paradoxa 151173·3
 Acacia melanoxylon 151386·7
 Acacia verniciflua 1515100
 Correa decumbens 1515100
 Prostanthera eurybioides 141071·4
 Platylobium obtusangulum 55100
 Eucalyptus sieberi 1313100
Batch 2
 Eucalyptus macrocarpa 7571·4
 Eucalyptus dalrympleana 1515100
 Eucalyptus viminalis var. viminalis99100
 Eucalyptus odorata 4375·0
 Acacia leiophylla 1414100
 Leptospermum myrsinoides 151280·0
 Pultenaea daphnoides 55100
 Pultenaea largiflorens 55100
 Pultenaea graveolens 88100
 Correa aemula 33100
 Eucalyptus sieberi 7571·4
Batch 3
 Acacia enterocarpa 4250·0
 Acacia pinguifolia 55100
 Acacia spooneri 1313100
 Allocasuarina robusta 1313100
 Brachyscome diversifolia 9555·6
 Banksia marginata 1515100
 Kunzea pomifera 1414100
 Eucalyptus sieberi 1515100

Susceptibility of 37 South Australian native plant species to infection by P. cinnamomi

The species tested varied in response to inoculation by P. cinnamomi. The majority of the inoculated plants of Eucalyptus baxteri, Pultenaea daphnoides, Pu. largiflorens, Pu. graveolens, Platylobium obtusangulum, Al. robusta, Al. muelleriana, Banksia marginata and K. pomifera died quickly after inoculation. By the end of the 16th week after inoculation, over 80% of the inoculated plants of these species had died, suggesting that they were highly susceptible (Table 2, Figs 1–3). Diseased plants of all these species exhibited sudden and severe wilt, followed by rapid plant death. At the time of death they showed severe root necrosis with almost complete loss of fine roots, and in many cases rot had girdled the collar region. Root samples from fine and lateral roots and the collar region consistently yielded P. cinnamomi (Table 2). A few control plants of these species also died during the period of observation. One plant each of E. baxteri, Pu. daphnoides, Pu. graveolens, two each of Pl. obtusangulum, B. marginata and K. pomifera, and one Al. robusta in batch 3 died. Root samples from these dead plants did not yield P. cinnamomi. Mortality of the inoculated plants was significantly greater than that of the corresponding non-inoculated controls (< 0·05) for all species except for Pl. obtusangulum and Pu. daphnoides (Table 2).

Individual plants of 15 species, namely Eucalyptus cladocalyx, E. viminalis var. viminalis, E. odorata, Leptospermum continentale, L. myrsinoides, L. juniperium, Acacia verniciflua, Allocasuarina verticillata, O. pannosa ssp. pannosa, Hakea rostrata, C. decumbens, C. aemula, C. calycina, Prostanthera eurybioides and Po. halmaturina ssp. halmaturina, varied in susceptibility to P. cinnamomi. A variable number of inoculated plants, ranging from one to 11 from each of these species, died of phytophthora dieback during the period of observation (Table 2). Responses varied from sudden death to slow decline. In the case of slow decline, leaves initially turned pale green and wilted and, in the case of Eucalyptus species, turned brown and abscissed. Diseased plants were weak with flaccid, pale green leaves. Death occurred weeks or months after the initial appearance of symptoms. However, a small number of plants of E. viminalis var. viminalis, C. aemula and H. rostrata showed sudden wilting and rapid death. Plants of these species showed varying severity of root rot, from lesions confined to a few spots along fine roots to extensive rot, suggesting they were moderately susceptible (Table 2). Phytophthora cinnamomi was regularly isolated from fine and lateral root samples that showed spot lesions and occasionally from the collar. In one plant of E. viminalis var. viminalis, P. cinnamomi was observed to have spread from one lateral root to a branch along one side of the stem, causing the infected branch to wilt and die. Some plants of each of these species remained alive and healthy until the end of experiment, although root samples and potting mix consistently yielded P. cinnamomi (Tables 2 and 3). A variable number of the non-inoculated control plants also died during the observation period, ranging from one plant each of E. viminalis var. viminalis, L. myrsinoides, Po. halmaturina ssp. halmaturina and O. pannosa ssp. pannosa to four plants of H. rostrata. Except for C. decumbens, there was no significant difference between mortality of the inoculated and non-inoculated plants within each species of this group (> 0·05).

Mortality of inoculated Pr. eurybioides, which was tested twice in UC mix, ranged from 46·7 (batch 1, inoculated in spring) to 73·3% (batch 6, winter). Plant mortality was 46·7% when tested in BioGro® in batch 7 (inoculated in winter). Allocasuarina robusta was tested twice in UC mix, with 80% mortality in the first test, inoculated in summer, and 20% in the second, in winter.

For six species, namely Acacia paradoxa, A. melanoxylon, A. spooneri, A. leiophylla, Eucalyptus microcarpa and E. dalrympleana, mortality of inoculated plants was very low (Table 2). Only one plant each of A. paradoxa, A. melanoxylon, A. spooneri and E. microcarpa, and two plants each of A. leiophylla and E. dalrympleana died and reisolation yielded P. cinnamomi. Root lesions, if any, did not develop into rot but remained confined to small localized lesions along fine roots from which P. cinnamomi was reisolated. The majority of plants of these species remained healthy for at least 11 weeks after inoculation, suggesting they were only slightly susceptible (Figs 1–3). Among the non-inoculated plants, only one plant of E. dalrympleana died. There was no significant difference in mortality between the inoculated and the non-inoculated plants for any of these species.

All inoculated plants of Acacia enterocarpa and A. pinguifolia remained healthy and continued to grow vigorously, reaching a height of 78·7 cm, an increase of 65 cm by the end of experiment. No chlorosis appeared in the phyllodes of either the inoculated or non-inoculated plants. Roots remained light-brown and healthy. However, root tissue from 50% and 40% of inoculated seedlings of A. enterocarpa and A. pinguifolia, respectively, yielded P. cinnamomi at the end of experiment, suggesting that these plants were resistant to phytophthora dieback. None of the control plants of these species died or yielded P. cinnamomi.

Brachyscome diversifolia was the only species from which P. cinnamomi was never reisolated from root tissue, suggesting that this species might not be a host of the pathogen (Table 4). Of the nine inoculated plants, two Br. diversifolia plants died during the study period, but P. cinnamomi was not reisolated from the roots. The remaining seven plants continued to grow without showing any noticeable dieback symptoms at the end of the experiment.

Table 4.   Response of 100- and 180-day-old seedlings of six South Australian native plant species 12 weeks after inoculation with Phytophthora cinnamomi
No.SpeciesAge of seedling (days)Mortality (%)aNo. inoculatedNo. deadNo. yielding P. cinnamomiMean weeks until death (range)Log-rank (Mantel–Cox) testbNo. controlsNo. controls deadc
  1. aPercentage mortality based on number of dead plants which yielded P. cinnamomi.

  2. bLog-rank (Mantel–Cox) was used to compare the survival curves of the inoculated and non-inoculated plants within the same species at  0·05 (S, significant; NS, not significant).

  3. c P. cinnamomi was not isolated from roots or potting mix from any of the non-inoculated controls.

  4. d E. sieberi was included as a susceptible control.

1 Eucalyptus viminalis var. viminalis18001500S51
10053·315885 (3–7) 51
2 Eucalyptus goniocalyx 1806·715118NS51
100015100 50
3 Eucalyptus cladocalyx 1806·7151111NS50
1006·7151111 50
4 Allocasuarina robusta 18040·015865·7 (5–7)NS50
10040·015966·2 (4–7) 50
5 Kunzea pomifera 18033·315656·8 (2–11)NS50
10026·715445·5 (3–12) 50
6 Acacia paradoxa 180015000NS50
1006·7152111 50
Eucalyptus sieberi d 10066·71511105·7 (3–11)S50

Results for Austrodanthonia carphoides, G. tabacina, Hakea rugosa and Or. eriopoda were inconclusive because all of the inoculated and non-inoculated plants died during the experiment.

Effect of seedling age on susceptibility to phytophthora dieback

Mortality of the susceptible control plants, E. sieberi, reached 66·7% 12 weeks after inoculation, which was significantly more than for the non-inoculated control (Table 4), indicating that the experimental conditions were conducive for P. cinnamomi to infect and kill plants.

Of the six species tested, seedling age influenced the response only of E. viminalis var. viminalis to infection by P. cinnamomi. Eight seedlings of E. viminalis var. viminalis inoculated when 100 days old died during the period of observation (12 weeks) whereas none inoculated when 180 days old died (significant difference, < 0·01). The response of E. goniocalyx, E. cladocalyx, Ac. paradoxa, Al. robusta and K. pomifera to inoculation with P. cinnamomi at 100 and 180 days did not differ. At 12 weeks after inoculation, mortality of Al. robusta was 40% and K. pomifera 26·7% to 33·3% while that for both E. goniocalyx and E. cladocalyx was 6·7%. Three control plants, one from E. goniocalyx at 180 days old and two from E. viminalis var. viminalis, one each at 100 and 180 days old, died during the experiment, but P. cinnamomi was not reisolated from the roots.

Discussion

The 37 species of South Australian native plants studied showed various degrees of susceptibility to phytophthora dieback. Of the 15 threatened or uncommon species tested, two were highly susceptible, six moderately susceptible, two slightly susceptible, three resistant and results for two species were inconclusive. Brachyscome diversifolia, an endangered perennial herb, was the only species not infected by P. cinnamomi. Of these 15 species, only Pugraveolens, from the provenance of Brisbane Ranges, Victoria, had been tested for susceptibility before and, likewise, found to be highly susceptible (Peters & Weste, 1997).

Of the 22 common species tested, seven were highly susceptible to the disease, nine moderately susceptible, four slightly susceptible and results from two species were inconclusive. The high susceptibility of E. baxteri to phytophthora dieback, which is consistent with a report by Podger & Batini (1971), may be a cause for concern because this species is abundant in the high rainfall areas of South Australia, namely southern parts of the Mount Lofty Ranges and Kangaroo Island, where P. cinnamomi is widespread (Williams et al., 2007).

Although Pl. obtusangulum and Pu. daphnoides were highly susceptible to phytophthora dieback, dieback of these species in their natural habitats in South Australia has not been reported. At Mount Bold Reservoir, Mount Lofty Ranges, where both species are common and P. cinnamomi has been isolated, mortality of these species was not evident and most of the dead plants were X. semiplana and Isopogon ceratophyllus (data not shown). The apparent differences in plant susceptibility between greenhouse and field may reflect differences in environmental and soil conditions. In the greenhouse, the plants were exposed to P. cinnamomi in conditions of temperature, moisture and medium conducive for infection and disease, while in the field environment, the conditions fluctuate widely.

Considerable variation in susceptibility among plants from different genera and among species (interspecific) from the same family was observed among the plants tested. Within the Fabaceae, species of Acacia tended to be resistant to the disease while all three Pultenaea species tested were highly susceptible. Also reported to be highly susceptible in greenhouse experiments were Pultenaea hibbertioides, Pu. paleacea, Pu. prostrata in Tasmania (Barker & Wardlaw, 1995) and Pu. subalpina in Victoria (Reiter et al., 2004). Within the Myrtaceae, responses among species of Eucalyptus to inoculation with P. cinnamomi varied widely, in that E. microcarpa and E. dalrympleana were resistant, E. viminalis var. viminalis and E. cladocalyx were moderately susceptible and E. baxteri was highly susceptible. This may reflect the genetic variability of Eucalyptus species that occupy different ecosystems. Con-generic and interspecific variation in susceptibility to phytophthora dieback among Australian native plant species is commonly reported in literature (Wills, 1993; Barker & Wardlaw, 1995; Shearer et al., 2004).

Phytophthora cinnamomi is generally considered to be a pathogen of woody perennials, with herbs, grasses and geophytes not affected (Wills, 1993; Shearer et al., 2004). In the present study, none of the herbaceous species tested, viz. Br. diversifolia, G. tabacina, Or. eriopoda and Au. carphoides were affected by the pathogen. Although most plants of G. tabacina, Or. eriopoda and Au. carphoides, both inoculated and non-inoculated, died, P. cinnamomi was isolated only from one inoculated plant of Or. eriopoda. Therefore, it cannot be concluded that phytophthora dieback was the cause of death. Barker & Wardlaw (1995) reported high mortality of the herbs Mitrasacme distylis, Stylidium despectum and S. perpusillum in the greenhouse; death may be due to the short-lived nature of these plants and, perhaps, the moist potting mix rather than P. cinnamomi.

Although herbs and orchids are unlikely to be affected, there is a need to assess the indirect impact of the disease on these species (Shearer et al., 2007). Of the 145 threatened South Australian native plant species listed by Velzeboer et al. (2005) as potentially vulnerable to infection by P. cinnamomi, 38% are trees or woody shrubs, 30% are herbs and 32% are orchids. Several populations of the woody species, such as Euphrasia collina ssp. osbonii, Sprengelia incarnata, Melaleuca squamea and Grevillea aquifolium, are located in P. cinnamomi -infested areas. Common overstorey species such as eucalypts and acacias were included in the study because death due to phytophthora dieback may affect understorey plants and hence alter the community structure and function (Weste, 1986). However, the impact on South Australian native herbs, grasses and geophytes remains to be determined.

The response of plant species to infection by P. cinnamomi is sometimes inconsistent even in the greenhouse. For example, Al. robusta was highly susceptible in the first test in UC mix, inoculated in summer, but moderately susceptible in the second. Whereas Pr. eurybioides was consistently deemed susceptible when tested twice in UC mix and once in BioGro®, mortality rates were 46·7% in BioGro® (batch 7), and 46·7 and 73·3% in UC mix (batches 1 and 6, respectively). Unfortunately, E. sieberi was not included as a susceptible control in batches 4–7. Differences between batches may arise from variation in experimental conditions, possibly seasonal, between tests. Slight variation in the time of inoculation (seasonal susceptibility), pH of the potting mix, temperature and watering regime can affect the growth and pathogenicity of P. cinnamomi (You et al., 1996; King & Dale, 2002). All these factors could cause variation in response to inoculation between tests. The variation in susceptibility between tests suggests that assigning a level of susceptibility to a species should be based on the results of multiple tests and not on a single experiment (Shearer et al., 2007). The greater consistency of response observed for E. sieberi may reflect the use of younger seedlings, which are highly susceptible (Smith & Marks, 1982).

Of the six species selected to assess the effect of seedling age on susceptibility to P. cinnamomi, only E. viminalis var. viminalis showed decreased susceptibility with increase in age. All species tested were fast-growing plants, and seedlings at 100 and 180 days old had mature root systems with dense lateral and feeder roots. The susceptibility to P. cinnamomi of plants with mature root systems and of younger seedlings having only seminal roots should be compared.

At the end of the experiment, root samples from 70% of all inoculated plants yielded P. cinnamomi. Of particular concern, two endangered plant species which are included in revegetation projects, Ac. enterocarpa and Ac. pinguifolia (Obst, 2005), were hosts of the pathogen. If symptomless plants with cryptic infection were planted in the natural ecosystem, they would provide a long-term source of inoculum for susceptible native plants in the vicinity. Therefore, ensuring that plant materials intended for revegetation are free from P. cinnamomi before being planted in the field is of the utmost importance. Furthermore, the use of Acacia spp. as companion plants to protect susceptible native species from infection by P. cinnamomi (D’Souza et al., 2004) should be carried out with caution.

Susceptible and endangered species such as Al. robusta and E. viminalis var. viminalis, which occur in small populations with restricted geographical distribution, are at risk from P. cinnamomi. For example, the total population of Al. robusta in South Australia is estimated to be 200 plants, all of which are confined to one location on the lower Fleurieu Peninsula (Obst, 2005). Similarly, E. viminalis var. viminalis, a rare species in South Australia, is confined to higher parts of the central Mount Lofty Ranges (Nicolle, 1997). About half of the populations of these species are located near to areas known to be infested with P. cinnamomi, putting them at a high risk of extinction if a chance infection occurs (Velzeboer et al., 2005).

This is the first detailed study of the susceptibility of South Australian native flora to phytophthora dieback and findings contribute to knowledge of the threat posed by the disease to South Australia. The information generated here may help in choosing species for conservation purposes. Natural populations of susceptible species identified in this study, particularly the endangered and rare species such as Al. robusta and C. aemula, should be located and tested for P. cinnamomi and, if at risk, appropriate measures, including ex situ conservation, should then be taken to protect them. For the common and susceptible species such as E. baxteri, research on selection of resistant plants and use of Acacia spp. with ability to protect native species from P. cinnamomi should be investigated to facilitate conservation and revegetation.

Acknowledgements

The authors thank the Australian Research Council for funding this research and the following for financial and logistical support: South Australian Department of Environment and Natural Resources, SA Water, Adelaide Hills Council, Adelaide and Mount Lofty Natural Resource Management Board, City of Tea Tree Gully Council, Department of Transport, Energy and Infrastructure, Forestry SA, Primary Industries and Resources South Australia Forestry, South Australian Murray Darling Basin Natural Resource Management Board and the University of Adelaide. We also thank the Sarawak State Government, Malaysia for sponsoring the PhD research of KHK; Dr N. M. Williams, D. White and Dr T. Burgess, Centre for Phytophthora Science and Management, Murdoch University, Western Australia for confirming identification of isolates 71a and SC4 and ITS sequencing; and Phillip Ainsley, Seed Conservation Centre, Department of Environment and Natural Resources for providing seeds and seedlings of threatened species.

Ancillary