The potential of five Western Australian native Acacia species for biological control of Phytophthora cinnamomi

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Australian Journal of Botany, 2004, 52, 267–279

The potential of five Western Australian native Acacia species for biological control of Phytophthora cinnamomi Nola K. D’SouzaA,D, Ian J. ColquhounB, Bryan L. ShearerA,C and Giles E. St. J. HardyA A

School of Biology and Biotechnology, Murdoch University, South St, Murdoch, WA 6150, Australia. Alcoa World Alumina Australia, Environmental Department, PO Box 242, Booragoon, WA 6953, Australia. C Department of Conservation and Land Management, Science Division, Locked Bag 104, Bentley Delivery Centre, WA 6983, Australia. D Corresponding author; email: [email protected]

B

Abstract. Five Acacia species native to Western Australia were assessed for their potential to protect the highly susceptible species Banksia grandis Wield from infection by the plant pathogen Phytophthora cinnamomi Rands. In a rehabilitated bauxite pit at Jarrahdale 55 km south-east of Perth and in a glasshouse trial, B. grandis planted either alone or with A. pulchella R.Br., A. urophylla Benth., A. extensa Lindl., A. lateriticola Maslin or A. drummondii Lindl., was soil inoculated with P. cinnamomi. It could only be shown that A. pulchella significantly protected B. grandis from P. cinnamomi infection in the rehabilitated bauxite pit trial up to 1 year after inoculation. This confirms the potential of this species for biological control of the pathogen in infested plant communities. The observed protection was not the result of a decrease in soil temperature or moisture. Protection was not emulated in a glasshouse trial where optimum environmental conditions favoured P. cinnamomi. Despite a delay in infection of B. grandis planted with Acacia spp., none of the five species definitively protected B. grandis from P. cinnamomi. However, in the glasshouse trial, A. pulchella, A. extensa, A. lateriticola and A. drummondii did significantly reduce the soil inoculum of P. cinnamomi, indicating a possible biological control effect on the pathogen. The mechanisms of biological control are discussed and the implications for management of rehabilitated bauxite mined areas and forests severely affected by P. cinnamomi are considered. BT03089 NeP.taoKle.nDtia’lSoufzAac ia speciesforbiolgicalconrtol

Introduction Phytophthora cinnamomi Rands is an important destructive pathogen in horticultural industries and native plant communities in many parts of the world. One of the highest impacts in Australia is in the jarrah forest and banksia-dominated woodlands of southern Western Australia (Podger 1972; Shearer and Tippett 1989). Within the south-west’s Stirling Range National Park, 85% of species in the dominant Proteaceae family are susceptible to P. cinnamomi (Wills 1993; Wills and Keighery 1994). Since its detection in Australia there has been much research into combating the threat of this pathogen (Podger and Batini 1971; Old 1979; Shearer and Tippett 1989; Marks and Smith 1991; Shearer and Smith 2000; Colquhoun and Hardy 2000). Chemical control, favoured in horticultural industries (Coffey 1991; Ribeiro and Linderman 1991), is impractical and uneconomic for effective control in native eucalypt forests. An exception is where conservation of endangered species that are susceptible to P. cinnamomi is a priority. This, coupled with environmental concerns and the possibility of ineffectiveness (Bailey and Coffey 1986; © CSIRO 2004

Cohen and Coffey 1986; Coffey 1991; Ribeiro and Linderman 1991; Fang and Tsao 1995), has led to an increased emphasis on finding non-chemical forms of control, including biological control (Linderman et al. 1983; Neumann and Marks 1990). Biological control of specific pests and pathogens, including P. cinnamomi (Hoitink et al. 1977; Cook and Baker 1983; Hardy and Sivasithamparam 1991; Fang and Tsao 1995; Downer et al. 2001), is an established practice in commercial and horticultural industries, but has not yet been established in native forests. Dense stands of legume species, in particular Acacia pulchella R.Br., have been investigated for biological control of P. cinnamomi in native Eucalyptus marginata Donn. (jarrah) forest of Western Australia. It was observed that the soil physical environment was less favourable to P. cinnamomi beneath stands of A. pulchella that germinated after uncontrolled forest fires (Shea et al. 1976). Sporulation by the pathogen was significantly suppressed in soil beneath A. pulchella compared with forest sites where the understorey was dominated by species within the family Proteaceae (Shea 10.1071/BT03089

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et al. 1978). In glasshouse trials, Shea et al. (1976) found that mortality of moderately susceptible E. marginata seedlings in pots was less when planted with A. pulchella than with Banksia grandis Wield, a highly susceptible species. The protection of B. grandis by A. pulchella has not been tested. The possibility of favouring regeneration of legumes by changing fire management practices was the most promising broad-scale potential for biological control of P. cinnamomi in a forest situation (Shea and Malajczuk 1977). Unfortunately, implementation of fires with the required intensity to break the dormancy of Acacia seed in the soil is hampered by the infrequency of weather conditions favourable for fires of this intensity. Even with the right conditions, these fires are hard to control over large areas and cause extensive scarring and wood damage to economically important eucalypt species (Burrows 1985, 1987). Furthermore, in some areas, manipulation of the understorey by fire may not be compatible with vulnerable communities of high conservation value. Also, the species composition of the leguminous understorey resulting after fire can be varied and patchy owing to site preferences, and although much is known about A. pulchella, little is known about the effects of other legume species on P. cinnamomi in a natural environment (Shearer and Tippett 1989). Is it possible that other native legumes have the same effect as A. pulchella and can also protect susceptible species against P. cinnamomi infection? Another promising potential for biological control of P. cinnamomi by manipulation of the understorey is in management practices already in place for rehabilitation of bauxite-mined areas in Western Australia. Bauxite mining is one of the largest operations within the jarrah forest of Western Australia. Minimising the spread and impact of P. cinnamomi is a major environmental objective achieved by an intensive management program (Colquhoun and Hardy 2000). The ability to modify the understorey seed mix ratio to promote a range of vegetation densities and species compositions alleviates some of the problems associated with manipulation of the understorey through fire management practices. The ability to influence a soil environment suppressive to P. cinnamomi for the rehabilitation of bauxite-mined pits in infested areas, offers another promising broad-scale potential for biological control of P. cinnamomi in severely affected forest. A field trial, within a rehabilitated bauxite pit, and a glasshouse trial were established to investigate the potential of five Western Australian native Acacia species to protect B. grandis—a species highly susceptible to P. cinnamomi infection. Materials and methods Rehabilitated bauxite pit trial site (field trial) The trial was located at Alcoa World Alumina Australia’s Jarrahdale mine 55 km south-east of Perth, Western Australia. It was established

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in August 1999 in a newly landscaped and rehabilitated P. cinnamomi-infested pit (Alcoa Grid Map Reference H3613). The pit was landscaped with stockpiled overburden and sandy gravel topsoil and seeded with a seed mix containing endemic species (Colquhoun and Hardy 2000). Jarrah (E. marginata) and marri (Corymbia calophylla R.Br. ex Lindley) were the dominant tree species in the seed mix in an 80:20 ratio. The pit was fertilised with diammonium phosphate (DAP) at 500 kg per hectare. The pit had a gradual slope into a bowl shape and the trial was positioned on the upper western face of the slope to ensure adequate drainage. Seedlings Banksia grandis was chosen for its high susceptibility to P. cinnamomi. Five of the most common Acacia species used in rehabilitation, A. pulchella, A. urophylla Benth., A. extensa Lindl., A. lateriticola Maslin and A. drummondii Lindl., were selected (Alcoa internal rehabilitation records) to determine their potential to protect B. grandis from P. cinnamomi in both the field trial and glasshouse trial. The seedlings were grown by Alcoa’s Marrinup Nursery (Dwellingup Western Australia) in ‘Wynelle’ pots (Plastic Injection Moulding, Ballarat, Victoria) with internal ribs to minimise root coiling (dimensions 50 × 50 mm top rim, 40 × 40 mm bottom and 122 mm depth). Experimental design A randomised block design was used for both trials, with 12 treatments and four replicates per treatment (Table 1). The mortality of B. grandis was assessed as the dependent variable, with Acacia spp. treatments and replicates as the independent variables. Planting procedure In the field trial, 16 B. grandis seedlings were planted 0.5 m apart in a 2 × 2-m grid. In Treatments B–F and H–L Acacia seedlings were planted around the B. grandis seedlings 0.5 m apart in a 2.5 × 2.5-m grid. All seedlings were approximately 6 months old at the time of planting in August 1999. Once planted, the seedlings were supplied with ~100 g of DAP fertiliser per plot, in addition to the initial fertiliser application of the whole pit, to aid in establishment. Each plot was positioned at least 1 m away from adjacent plots. The seedlings were allowed to establish for 10 months before inoculation of the soil with P. cinnamomi. During this time any other plant species that emerged from seed within the plots and within a 1m radius were manually removed. In the glasshouse trial eight B. grandis seedlings were planted 10 cm apart in a 10 × 30 cm grid in black PVC No. 10 Crate ‘Nally’ bins Table 1. Treatment A B C D E F G H I J K L

Treatments used in the rehabilitated bauxite pit and glasshouse trials Species planted Banksia grandis B. grandis and Acacia pulchella B. grandis and A. urophylla B. grandis and A. extensa B. grandis and A. lateriticola B. grandis and A. drummondii B. grandis B. grandis and A. pulchella B. grandis and A. urophylla B. grandis and A. extensa B. grandis and A. lateriticola B. grandis and A. drummondii

Soil inoculated with Phytophthora cinnamomi Yes Yes Yes Yes Yes Yes No No No No No No

Potential of Acacia species for biological control

(inside dimensions 597 × 362 × 266 mm, 52 L capacity (Product IH051, Silverlock and Co. Pty Ltd, Canning Vale, WA). Twenty-four 100-mm holes were drilled through the bottom of each bin to allow adequate drainage. In Treatments B–F and H–L, Acacia seedlings were planted around the B. grandis seedlings 10 cm apart in a 20 × 40-cm grid. The seedlings were approximately 1 year old when planted. The bins were filled up to 230-mm depth with 2-year-old stockpiled, screened, P. cinnamomi-free topsoil supplied from Alcoa’s Huntly mine. The screening process concentrates the topsoil seed bank by removing large debris and gravel before spreading it back onto mined pits for rehabilitation. Approximately 50 g of granular DAP fertiliser was supplied by hand to each treatment to aid establishment. After planting in the bins screw-capped 10-mL plastic tubes (12 cm long, 1 cm diameter) (Sarstedt, South Australia, Item 60 9921 819) were pushed into the soil at each inoculation point equidistant between B. grandis seedlings. This enabled inoculum plugs to be placed into the holes without damaging roots when the tubes were removed. The bins were hand watered to container capacity approximately every second day to maintain soil moisture. Hand watering ensured that no splash occurred between bins that could have caused contamination of control treatments. Acacia density Grazing pressure greatly affected the establishment of some of the Acacia spp. seedlings. This was compounded by drought during the first summer so that 6 months after planting a total of 69% of A. urophylla, 56% of A. extensa, 39% of A. drummondii, 23% of A. lateriticola and 8% of A. pulchella seedlings across plots had died. Replanting occurred in March 2000 but there were insufficient seedlings available to replace all deaths. Consequently, 29% (58) of A. urophylla, 16% (32) of A. extensa, 4% (8) of A. drummondii, 1% (2) of A. lateriticola and 4% (8) of A. pulchella seedlings were not replaced across plots prior to inoculation. This reduced the density of Acacia spp. from 6.25 plants m–2 to 4.4, 5.25, 6, 6.2 and 6 plants m–2, respectively, prior to soil inoculation in June 2000. From November 2000 to April 2001 drought stress affected many of the surviving Acacia spp.—74% (147) of A. drummondii, 62% (124) of A. lateriticola, 55% (110) of A. urophylla, 42% (84) of A. extensa and 13% (25) of A. pulchella had died across plots by the end of the experiment. This resulted in final densities of 1.7, 2.4, 2.8, 3.6 and 5.5 plants m–2, respectively. Preparation of Banksia plug inoculum Young green stems of B. grandis were collected from remnant dieback-free bushland along Jarrahdale Road, (55 km south of Perth). Leaves were removed on site and the stems stored in a bucket of water overnight in the laboratory. Stems were cut with a bandsaw into plugs 2.5 cm long and 1–2 cm in diameter. The plugs were rinsed to remove sawdust and insects and then soaked overnight in distilled water. Plugs were placed into 2-L conical flasks (120 plugs per flask) with ~1 cm of distilled water in the bottom of the flask. The necks of the flasks were plugged with cotton wool and covered with two layers of aluminium foil. They were then autoclaved at 138 kPa for 30 min at 121°C and again 24 and 96 h later for 20 min. Four known pathogenic isolates of P. cinnamomi originally isolated from the Jarrahdale mine (MP97-12 and MP94, highly pathogenic in jarrah; and MP127 and MP97-7, moderately pathogenic in jarrah) (Hüberli et al. 2001), were grown separately for 1 week on pea agar plates (10% w/v frozen green peas, Difco Bacto Agar) in 90-mm diameter plastic Petri dishes and then cut into 1-cm2 pieces and aseptically transferred to the flasks containing the autoclaved plugs. Two plates of colonised agar were used per flask and the flask was gently shaken to ensure even distribution of the colonised agar squares.

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The cotton wool and aluminium foil plugs were replaced and the flasks were then sealed with plastic kitchen wrap to prevent contamination. The flasks were incubated at 24°C in constant light for a minimum of 4 weeks before use. Inoculation In the field trial at the beginning of June 2000, one P. cinnamomi-infested plug of each isolate was buried 10–20 cm below the surface at four equidistant points, 0.25 m away from each B. grandis plant in Treatments A–F. To minimise root damage, holes were made by hammering a spiked, wooden peg, 5 cm diameter, into the ground to the required depth prior to placing the plug into the hole. In the glasshouse trial, seedlings were allowed to establish for 5 months before inoculation. The plastic tubes were removed and replaced with plugs of the same four isolates used in the field trial. Each Banksia seedling was exposed to each of the four P. cinnamomi isolates. Inoculum viability In order to confirm inoculum viability in the field trial, two plugs of the isolate corresponding to the replicate number of the plot (e.g. Isolate 1—MP94 in all number 1 replicate plots) were buried in 5-cm2 nylon-mesh bags attached to nylon string and a plastic tag that remained above the surface (Bunny 1996). In September 2000, 3 months after burial, the plugs were retrieved to determine if P. cinnamomi remained viable when the plugs were surface sterilised and plated onto NARPH Phytophthora-selective agar (Hüberli et al. 2000). The inoculum in all 96 plugs was viable and consequently it was considered unnecessary to repeat the inoculation process. Harvest The field trial was monitored monthly for dead or dying B. grandis seedlings during the autumn and winter months and fortnightly during the spring and summer months for the duration of the experiment (June 2000–May 2001). The glasshouse trial was monitored three times a week for 12 weeks. The roots and collar region of dead or dying B. grandis plants in each trial were processed to determine if P. cinnamomi had invaded the tissues. Approximately 5-cm lengths of the roots and collars were surface-sterilised in 70% ethanol for 30 s, then washed in three rinses of distilled water and dried on blotting paper. The root and collar segments were aseptically cut longitudinally to expose the cortex. The exposed tissue was directly plated by using sterile forceps onto NARPH Phytophthora-selective agar (Hüberli et al. 2000). The plates were incubated in constant light at 24°C for 72 h and then examined under a light microscope for the presence of P. cinnamomi hyphae. Measurement of physical and chemical soil properties Soil physical and chemical properties were determined at both the field site and from the screened topsoil used in the glasshouse prior to planting. Four random samples from across the field site were obtained by using a 45-mm diameter auger (0–20 cm depth) and four samples were obtained from the screened topsoil by using a hand trowel. The samples were mixed and soil was oven-dried for 24 h at 105°C. Physical characteristics were determined by the particle size hydrometer method (Day 1965; Gee and Bauder 1986). Conductivity, pH and total dissolved salts were determined according to Rayment and Higginson (1992). Total nitrogen was determined by the semimicro-Kjeldahl steam-distillation method (McKenzie and Wallace 1954). Potassium was determined by the bicarbonate extractable method (Rayment and Higginson 1992). Available phosphorus was determined by colourimetric determination (Murphy and Riley 1962; Watanabe and Olsen 1965) and organic carbon was determined by the rapid titration method (Piper 1942). These processes were repeated on

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samples taken from each replicate of each treatment 1 year after planting the field trial and at the end of the glasshouse trial. Measurement of soil temperature In the field trial, the ambient temperature beneath a shrub canopy was measured with one thermistor every 4 h from the time of inoculation by using a Unidata Starlog Data Logging System version 2.21. The soil temperature at 20-cm depth was also measured every 4 h with six thermistors in six representative plots of each of the control treatments. Soil temperature was compared among treatments. Thermistors were also used in the glasshouse to monitor soil and ambient temperatures every 2 h, to confirm that the temperature was conducive to infection by P. cinnamomi. One thermistor recorded ambient temperature and two thermistors buried at 20-cm depth in two Nally bins on the north and south side of the glasshouse recorded soil temperature. The ambient temperature thermistor malfunctioned, but the soil thermistors showed that average daily and hence monthly soil temperatures remained within a range of 15–25°C, which was conducive to P. cinnamomi infection. Measurement of soil moisture at the field trial Soil moisture was determined from every field plot once in June 2000 at the time of inoculation and then monthly from September 2000 until May 2001. Each sample was obtained by using a 45-mm-diameter auger (0–20 cm depth). Soils were weighed wet and then oven-dried for 24 h at 105°C. The soils were weighed again then sifted through a 2-mm sieve and the portion with >2-mm particle size was also weighed. This portion, consisting of rocks and gravel, was assumed to hold no moisture so its weight was subtracted from the wet weight and moisture loss was determined to have come from the portion of 0.05) different from non-inoculated plots of B. grandis planted alone or from the corresponding non-inoculated plots of B. grandis planted with A. pulchella (Fig. 1). Mortality due to P. cinnamomi infection of B. grandis planted with A. lateriticola or A. drummondii was not significantly (P > 0.05) different from mortality of soil-inoculated B. grandis growing alone (Fig. 1). These two species were not able to protect B. grandis from P. cinnamomi infection. The densities of A. urophylla and A. extensa were reduced to 4.4 and 5.2 plants m–2, respectively, prior to soil 100 90 80

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Fig. 1. Mortality of Banksia grandis in non-inoculated control plots (hollow columns) and in plots inoculated with Phytophthora cinnamomi (solid columns) when planted with five different Acacia species in a rehabilitated bauxite pit. Bars represent standard errors of the mean. Treatments A and G = B. grandis planted alone, B and H = B. grandis planted with Acacia pulchella, C and I = B. grandis planted with A. urophylla, D and J = B. grandis planted with A. extensa, E and K = B. grandis planted with A. lateriticola and F and L = B. grandis planted with A. drummondii.

Potential of Acacia species for biological control

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Fig. 2. Mortality of Banksia grandis over time in non-inoculated (dotted line) control plots and in plots inoculated (solid line) with Phytophthora cinnamomi when planted with five different Acacia species in a rehabilitated bauxite pit. Trial was inoculated in June 2000. B. grandis planted alone (×), B. grandis planted with Acacia pulchella (䉱, 䉭), B. grandis planted with A. urophylla (䉬, 䉫), B. grandis planted with A. extensa (+), B. grandis planted with A. lateriticola (䊏, 䊐) and B. grandis planted with A. drummondii (䊉, 䊊).

inoculation. This compromised the efficacy of these Acacia species. Although mortality due to P. cinnamomi infection of B. grandis planted with A. urophylla or A. extensa was not significantly (P > 0.05) different from mortality of soil-inoculated B. grandis growing alone, the effectiveness of these two species could not be determined. Despite the compromise of control plots by the resident P. cinnamomi population there was still significantly (P < 0.05) more disease over time in inoculated than in non-inoculated plots, except in the A. pulchella plot, owing to the addition of the extra inoculum (Fig. 2). Mortality of B. grandis rose more sharply from November 2000 to January 2001 in inoculated plots than in non-inoculated plots, except in the inoculated A. pulchella plot. Physical and chemical measurements Soil properties There was a significant (P < 0.05) increase in total nitrogen and organic carbon in every treatment compared with the pre-trial analysis measured before planting (Table 2). There was no significant (P > 0.05) difference in potassium or phosphorus levels compared with the pre-trial analysis. There were some significant (P < 0.05) differences for conductivity, total soluble salts and pH compared with the pre-trial analysis but there was no single consistent treatment effect that contributed to the protection of B. grandis by A. pulchella (Table 2).

The only significant (P < 0.05) difference in soil moisture between treatments occurred in November 2000, February 2001 and April 2001. These treatment differences were not consistent. In November 2000, non-inoculated plots of B. grandis growing with A. pulchella (H) had the highest mean soil moisture (16%) and were significantly (P < 0.05) greater than all other treatments except Treatment I—non-inoculated plots of B. grandis planted with A. urophylla (13.5%) (Fig. 3). In February 2001, the difference between the highest and lowest mean soil moisture was only 3% (Fig. 3). In April 2001, soil inoculated plots with B. grandis and A. drummondii (F) had a significantly (P < 0.05) higher mean soil moisture (9.9%) than all other treatments in the same month (Fig. 3).

Temperature and rainfall Significant (P < 0.05) soil temperature differences were observed between treatments from July 2000 to November 2000 and again in February 2001. Rainfall events and soil temperature conditions above 15°C that are favourable for P. cinnamomi infection are most likely to coincide in spring (Shea and Malajczuk 1977). During spring from September to November 2000, a temperature depression below 15°C was observed at some point within the plots containing A. pulchella, A. lateriticola and A. drummondii but not A. urophylla or A. extensa, when compared with plots of B. grandis planted alone where soil temperatures were above 15°C. Rainfall occurred on 13 days during September but only one rainfall event coincided with soil temperatures above 15°C in this month (Fig. 4). The average monthly soil temperature in September for the A. pulchella plot was not significantly (P > 0.05) different from B. grandis planted alone. However, the average daily temperature was depressed below 15°C in this plot and the A. lateriticola and A. drummondii plots during 21–26 September (Fig. 4). Rain was recorded on 4 days in October and in each case was less than 5mm (Fig. 5). All of these rainfall events coincided with soil temperatures above 15°C in all plots, except 6 October where soil temperatures in A. pulchella, A. lateriticola and A. drummondii plots were below 15°C. Soil temperature of plots containing A. urophylla also became significantly depressed from 14 to 31 October compared with B. grandis planted alone; however, this was not below 15°C. By 15 October the temperature in all plots was consistently higher than 15°C. In November rain occurred on 5 days and soil temperatures in all plots were consistently higher than 15°C (Fig. 6). By the end of November soil temperatures in each plot were no longer significantly (P > 0.05) different from each other.

B. grandis and A. drummondii

B. grandis and A. lateriticola

B. grandis and A. extensa

B. grandis and A. urophylla

B. grandis and A. pulchella

Pre-trial soil analysis B. grandis alone

A G B H C I D J E K F L

1.8 ± 0.1a 2.7 ± 0.3abcd 1.9 ± 0.3a 3.1 ± 0.6bcd 4.1 ± 0.8d 2.4 ± 0.1abc 2.6 ± 0.1abcd 3.0 ± 0.6bcd 3.0 ± 0.5bcd 2.7 ± 0.1bcd 3.9 ± 1.3cd 3.3 ± 0.1bcd 2.2 ± 0.2ab

Conductivity (mS m–1) 5.4 ± 0.05ab 5.5 ± 0.2a 5.1 ± 0.1cdef 5.3 ± 0.04abcd 5.2 ± 0.05cdb 5.1 ± 0.04cdef 4.9 ± 0.2f 5.0 ± 0.05edf 4.9 ± 0.1ef 5.1 ± 0.04cde 5.3 ± 0.1abc 5.2 ± 0.04bcd 5.3 ± 0.02abcd

pH (dist. H2O) 0.03 ± 0.002ab 0.04 ± 0.01bcd 0.03 ± 0.01a 0.1 ± 0.1cd 0.1 ± 0.02d 0.04 ± 0.003abcd 0.04 ± 0.002cd 0.05 ± 0.01cd 0.05 ± 0.01cd 0.05 ± 0.001cd 0.1 ± 0.03cd 0.1 ± 0.003cd 0.03 ± 0.005abc

Total soluble salts (g kg–1) 3.4 ± 0.7ab 1.8 ± 0.7b 4.5 ± 1.1b 1.8 ± 0.7b 12.4 ± 5.2a 2.6 ± 0.7b 4.9 ± 1.6b 3.4 ± 1.0ab 4.0 ± 2.0ab 3.1 ± 1.6ab 3.0 ± 0.8ab 5.3 ± 2.0ab 1.8 ± 1.0b

Phosphorus (HCO3, µg g–1) 36.4 ± 6.8ab 22.8 ± 6.7b 20.9 ± 4.0b 27.0 ± 8.2ab 41.4 ± 8.6a 19.3 ± 1.6b 22.6 ± 2.1ab 22.9 ± 2.3ab 24.6 ± 4.5ab 28.0 ± 4.0ab 40.3 ± 18.8ab 39.7 ± 4.6a 19.3 ± 1.7b

Potassium (HCO3, µg g–1)

0.01 ± 0.001a 1.2 ± 0.3c 0.9 ± 0.3b 1.5 ± 0.3c 1.5 ± 0.1c 1.0 ± 0.2bc 1.3 ± 0.2c 1.2 ± 0.05c 1.5 ± 0.2c 1.4 ± 0.1c 1.3 ± 0.1c 1.5 ± 0.3c 1.1 ± 0.1c

Organic carbon (%)

0.00 ± 0.00a 0.1 ± 0.01bcd 0.05 ± 0.01b 0.1 ± 0.01cd 0.1 ± 0.003d 0.05 ± 0.005bc 0.1 ± 0.005bcd 0.1 ± 0.001cd 0.1 ± 0.01cd 0.1 ± 0.001cd 0.1 ± 0.001cd 0.1 ± 0.01cd 0.1 ± 0.002bcd

Total nitrogen (%)

Table 2. Rehabilitated field trial: effect on physical and chemical soil properties of different Acacia spp. treatments compared with the pre-trial analysis Treatments A–F were inoculated with Phytophthora cinnamomi, G–L were not inoculated. Results (± standard error) with the same letter are not significantly different (P > 0.05)

272 Australian Journal of Botany N. K. D’Souza et al.

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Fig. 3. Average soil moisture of rehabilitated bauxite pit trial plots in months where there was a significant difference between treatments. Bars represent standard errors of the mean. Treatments A and G = Banksia grandis planted alone, B and H = B. grandis planted with Acacia pulchella, C and I = B. grandis planted with A. urophylla, D and J = B. grandis planted with A. extensa, E and K = B. grandis planted with A. lateriticola and F and L = B. grandis planted with A. drummondii. A–F inoculated, G–L non-inoculated.

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Fig. 5. Recorded rainfall and soil temperature in representative treatment plots during October 2000 in the rehabilitated bauxite pit trial. Soil temperature was consistently lower in plots planted with Acacia pulchella, A. lateriticola, A. drummondii and A. urophylla than with Banksia grandis (Bg) alone. All soil temperatures in different representative treatment plots were consistently higher than 15°C in the latter half of the month.

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Fig. 4. Recorded rainfall and soil temperature in representative treatment plots during September 2000 in the rehabilitated bauxite pit trial. A temperature depression below 15°C was observed in the Acacia pulchella, A. lateriticola and A. drummondii plots towards the end of the month compared with Banksia grandis (Bg) alone.

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N o 3- v-0 N 0 o 5- v-0 N 0 o 7- v-0 N 0 o 9- v-0 N 0 11 ov-0 -N 0 13 ov-N 00 15 ov-N 00 17 ov-N 00 19 ov-N 00 21 ov-N 00 23 ov-N 00 25 ov-N 00 27 ov-N 00 29 ov-N 00 ov -0 0

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Fig. 6. Recorded rainfall and soil temperature in representative treatment plots during November 2000 in the rehabilitated bauxite pit trial. All soil temperatures in different representative treatment plots were consistently higher than 15°C and do not significantly (P > 0.05) differ towards the latter half of the month. Bg, Banksia grandis; A, Acacia spp.

Glasshouse trial Treatment comparisons In the glasshouse trial all of the Acacia species significantly (P < 0.05) delayed the infection of B. grandis by P. cinnamomi for 7 weeks (Fig. 7). Death of B. grandis seedlings were first observed in inoculated treatments of B. grandis planted alone and in treatments of B. grandis planted with A. drummondii 4 weeks after soil inoculation. Deaths of B. grandis in all other treatments were first recorded in Week 5. There was significantly (P < 0.05) lower mortality of B. grandis in all inoculated Acacia spp. treatments

compared with inoculated treatments of B. grandis planted alone in Week 5 and this continued into Weeks 6 and 7 (Fig. 7). However, all inoculated treatments also had significantly (P < 0.05) higher mortality than the non-inoculated control treatments during Weeks 5–7 (Fig. 7). After 12 weeks, mortality of soil-inoculated B. grandis planted with A. urophylla, A. extensa and A. drummondii was not significantly (P > 0.05) different from soil-inoculated treatments of B. grandis planted alone. Mortality of soil-inoculated B. grandis planted with A. pulchella and

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Fig. 7. Mean mortality of Banksia grandis in each treatment resulting from Phytophthora cinnamomi infection over time in weeks after soil inoculation of a glasshouse trial. Bars represent standard errors of the mean. Treatments A–F inoculated; Treatments G–L non-inoculated. Treatments A and G = B. grandis planted alone, B and H = B. grandis planted with Acacia pulchella, C and I = B. grandis planted with A. urophylla, D and J = B. grandis planted with A. extensa, E and K = B. grandis planted with A. lateriticola and F and L = B. grandis planted with A. drummondii.

A. lateriticola was significantly (P < 0.05) lower than soil-inoculated B. grandis planted alone. However, there was still greater than 50% mortality attributed to P. cinnamomi in these two treatments and they were significantly (P < 0.05) different from the corresponding non-inoculated controls (Fig. 7). Despite the lower mortality of B grandis seedlings observed in these two treatments, it could not be determined if the remaining seedlings were protected from infection

because they died as a result of infection by another fungal pathogen. Inoculum potential of P. cinnamomi The planting of A. pulchella, A. extensa, A. lateriticola and A. drummondii with B. grandis in the glasshouse, significantly (P < 0.05) reduced the inoculum potential of P. cinnamomi in soil compared with B. grandis planted alone

Average colony forming units per gram of dry soil

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18 16 14 12 10 8 6 4 2 0

A

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Fig. 8. Phytophthora cinnamomi inoculum potential (colony-forming units per gram dry weight of soil) in soil planted with Banksia grandis alone (A) or with Acacia pulchella (B), A. urophylla (C), A. extensa (D), A. lateriticola (E) or A. drummondii (F). Bars represent standard errors of the mean.

(Fig. 8). A. urophylla had no effect on P. cinnamomi inoculum potential (Fig. 8). Physical and chemical measurements Soil properties Levels of all the soil properties measured were variable between treatments but there was no single consistent treatment effect (Table 3). Compared with the pre-trial analysis, there was a significant (P < 0.05) increase in total nitrogen, organic carbon, phosphorus, conductivity and total soluble salts in all treatments. There was a significant (P < 0.05) decrease in potassium levels in all treatments except inoculated B. grandis planted alone. There was a significant (P < 0.05) decrease in pH from the pre-trial analysis associated with inoculated treatments of A. pulchella planted with B. grandis (Treatment B). However, this was not significantly (P > 0.05) different from non-inoculated treatments of B. grandis planted alone and the decrease was not observed in the corresponding non-inoculated A. pulchella treatment (Treatment H). All other treatments did not significantly (P > 0.05) affect pH compared with the pre-trial analysis. Discussion Acacia pulchella was able to protect Banksia grandis against Phytophthora cinnamomi infection for over a year in a newly rehabilitated bauxite pit. This supports previous observations that A. pulchella can protect another susceptible species (Eucalyptus marginata) from P. cinnamomi infection in pot trials (Shea et al. 1976; Shea and Malajczuk 1977). The reduced density after replanting but before inoculation, from 6.25 to 4.4 plants m–2 for A. urophylla, severely compromised the efficacy of this species. To a lesser extent this was also the case for A. extensa, which was reduced to 5.25 plants m–2 before inoculation. It could not be

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determined if A. urophylla and A. extensa were effective at protecting B. grandis from infection by P. cinnamomi. Although the densities of A. drummondii and A. lateriticola were also severely compromised by the end of the trial, to 1.7 and 2.4 plants m–2, respectively, their densities were similar to that of A. pulchella (6 plants m–2) at the point before soil inoculation. The opportunity for P. cinnamomi to infect B. grandis plants during spring was not prevented by A. lateriticola and A. drummondii before they were affected by drought. A. lateriticola and A. drummondii were not able to protect B. grandis from P. cinnamomi infection. The level of protection by A. pulchella in the field trial was not emulated in the glasshouse trial, where optimum temperature and moisture conditions favoured P. cinnamomi. However, mortality of B. grandis due to infection by P. cinnamomi was significantly delayed for up to 7 weeks by all of the Acacia species tested. Previously, Smith and Marks (1983) also reported that seven out of nine Acacia species delayed infection of Eucalyptus sieberi by P. cinnamomi for up to 21 days (3 weeks). Although mortality of B. grandis in A. pulchella and A. extensa treatments was significantly lower that of than B. grandis planted alone after 12 weeks, none of the B. grandis seedlings survived in the long term. Significant mortality was observed in these treatments compared with the corresponding non-inoculated treatments. Hence, none of the Acacia species definitively protected B. grandis from infection in the glasshouse. The observed delay in mortality may be a result of the physical presence of the Acacia roots in the soil. Zoospores are chemotactically attracted to roots of resistant species as well as susceptible species (Malajczuk et al. 1977; Tippett and Malajczuk 1979). Zoospores attracted to the Acacia roots would have resulted in fewer zoospores available to infect the B. grandis roots, hence delaying mortality. Protection of E. marginata from P. cinnamomi infection by A. pulchella has previously been shown to be a result of reduced inoculum in the soil (Shea et al. 1976, 1978; Shea and Malajczuk 1977). In the present glasshouse trial, significantly lower soil inoculum potential was measured in four of the five Acacia spp. treatments, including A. pulchella. A. urophylla did not reduce the soil inoculum level but it still delayed mortality of B. grandis. Although the presence of the other Acacia spp. either directly or indirectly resulted in lower soil inoculum potential of P. cinnamomi, this did not prevent seedling infection in an environment conducive to the pathogen. The delay in mortality does not seem to be a direct result of reduced inoculum in the soil. Time constraints meant that soil inoculum was not measured in the field trial, so the suppression of P. cinnamomi sporulation could not be determined. The environmental conditions in the glasshouse were ideal for P. cinnamomi sporulation and infection; consequently, it is difficult to extrapolate from these results to a natural

B. grandis and A. drummondii

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2.8 + 0.1a 4.9 ± 0.6b 5.7 ± 0.4bcd 5.6 ± 1.0bc 5.8 ± 0.3bcd 8.6 ± 0.7e 6.9 ± 0.4cde 7.7 ± 0.6de 7.2 ± 0.9cde 6.2 ± 0.5bcd 6.2 ± 0.4bcd 6.8 ± 1.1bcde 7.8 ± 1.4de

Conductivity (mS m–1) 5.6 ± 0.05a 5.5 ± 0.1abc 5.3 ± 0.03cd 5.2 ± 0.04d 5.5 ± 0.02abc 5.4 ± 0.1bc 5.5 ± 0.05ab 5.4 ± 0.1abc 5.5 ± 0.04abc 5.4 ± 0.1abc 5.6 ± 0.03a 5.4 ± 0.1abc 5.5 ± 0.1ab

pH (dist. H2O) 0.04 ± 0.002a 0.1 ± 0.01b 0.1 ± 0.01bcd 0.1 ± 0.02bc 0.1 ± 0.01bcd 0.2 ± 0.02e 0.1 ± 0.01cde 0.2 ± 0.01de 0.1 ± 0.02cde 0.1 ± 0.01bcde 0.1 ± 0.01bcde 0.1 ± 0.02bcde 0.2 ± 0.03de

Total soluble salts (g kg–1) 0.05)

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ecosystem where conditions may not be as favourable. A reduction in inoculum in a natural ecosystem when combined with less conducive soil conditions for disease development may result in reduced disease incidence, as was observed for plots with A. pulchella in the field trial. These conclusions need to be confirmed and should be quantified in future rehabilitated bauxite pit and forest trials. In previous studies, four main theories were proposed to explain how A. pulchella suppressed P. cinnamomi soil inoculum and protected susceptible plant species: (i)

Physical alterations to the soil environment that disfavour the pathogen (Shea and Malajczuk 1977; Smith and Marks 1983; Smith et al. 1989). (ii) Changes in soil chemical composition (Nesbitt et al. 1980). (iii) Microbial interactions antagonistic to the pathogen (Broadbent et al. 1971; Shea and Malajczuk 1977; Murray et al. 1985; Murray 1987). (iv) Direct or indirect action of plant root exudates on the pathogen (Krupa and Nylund 1972; Whitfield et al. 1981). The present field observations showed that alterations to the physical factors of soil temperature and moisture or chemical composition were not responsible for the protection of B. grandis by A. pulchella. Shea and Malajczuk (1977) suggested that the dense canopy and litter layer of stands of A. pulchella decreased soil temperature in spring below a critical temperature of 15°C and concluded that this prevented the coincidence of favourable soil moisture and temperature conditions that are required for P. cinnamomi sporulation when compared to open forest. Shea and Malajczuk’s (1977) observation was presented as a single week’s temperature regime in spring; the month was not stated. In the present study, measurements throughout spring supported that, except for A. extensa, the other Acacia species did depress the soil temperature when compared with plots of B. grandis without Acacia species. However, these temperature depressions fluctuated below 15°C from September through to 15 October 2000. After this date, during the latter half of spring, the soil temperature was consistently above 15°C in all plots. Rain was recorded on 7 of the 46 remaining days of spring, which could have induced sporulation. Furthermore, the temperature depression was not confined to soil beneath A. pulchella. There was also a temperature depression below 15°C beneath A. lateriticola and A. drummondii plots and these species did not protect B. grandis from infection. These data indicate that a decrease in soil temperature is unlikely to be responsible for protection of B. grandis by A. pulchella. Shea and Malajczuk (1977) also surmised that the dense A. pulchella canopy intercepted rainfall, preventing soil rewetting during summer and autumn. Smith and Marks (1983) determined that a reduction in soil moisture was one

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of the mechanisms involved in protection of E. sieberi by Acacia spp. in pot trials, but that this occurred via increased transpiration by the Acacia seedlings. They speculated that Acacia plants in the field were capable of transpiring substantial quantities of soil water and could protect susceptible species by reducing the soil moisture matric potential below the optimal level for infection. Smith et al. (1989) confirmed that soils were drier under Acacia canopies in a forest environment owing to increased transpiration. However, protection was not provided to susceptible species in this instance despite the reduced soil moisture (Smith et al. 1989). The findings of the current study further indicate there was no consistent Acacia treatment effect on soil moisture to support the proposition that decreased soil water content, which might result from either interception of rainfall or transpiration by dense canopies of A. pulchella, can protect B. grandis. Nesbitt et al. (1980) observed that zoospore release was reduced in both sterile and non-sterile leachates of some suppressive soils and concluded there may be some chemical property that prevents zoospore release or differentiation. The soil chemical and nutrient characteristics measured in the present field trial were not significantly influenced by the A. pulchella treatment that protected B. grandis when compared with the other Acacia treatments that did not protect B. grandis. Similarly in the present glasshouse trial, there were no obvious treatment differences in chemical and nutrient levels that could have caused a delay in infection in all the treatments. It is possible that other chemicals or nutrients not measured in the present trials could have caused the observed effects. It has previously been shown that microorganisms associated with roots of A. pulchella can cause hyphal lysis and abortive sporangia of P. cinnamomi, resulting in reduced sporulation (Shea and Malajczuk 1977). Of the bacterial isolates from the rhizosphere of A. pulchella, 45% contained bacteria that were antagonistic to P. cinnamomi compared with 18% of isolates from B. grandis (Shea and Malajczuk 1977). Recently, biological control of P. cinnamomi in organic matter substrates has been attributed to soil enzyme activities. Cellulase and laminarinase produced by litter decay fungi declined with distance away from mulched areas with higher microbial activity, while the prevalence of Phytophthora spp. increased (Downer et al. 2001). Whitfield et al. (1981) showed that A. pulchella root exudates consist of volatile organic compounds that inhibit hyphal growth, suppress production of sporangia and reduce germination of zoospores of P. cinnamomi. These volatile exudates are probably produced by many legumes and may play a part in protection, not only by direct chemical interference but also by the indirect attraction of antagonistic microorganisms to the rhizosphere (Murray et al. 1985). Time constraints during this study prevented a thorough examination of the microbiological or root exudate

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mechanisms that have been suggested previously. Either mechanism might be involved in the observed protection of B. grandis by A. pulchella in the field trial or the suppression of P. cinnamomi soil inoculum by four of the five Acacia spp. in the glasshouse trial. Comparing the type and amount of microorganisms associated with A. pulchella and other Acacia species could indicate if they were involved in the protection of B. grandis. So too would a comparison of root exudates between A. pulchella and other Acacia spp. This study confirms the potential of A. pulchella for the biological control of P. cinnamomi in infested plant communities. In addition A. extensa, A. lateriticola and A. drummondii significantly reduced the soil inoculum of P. cinnamomi in the glasshouse trial, indicating a possible biological control effect on P. cinnamomi. Although these three species showed no control in the present rehabilitated bauxite pit trial, their direct action on the pathogen in the pit soil was not determined and their potential requires further investigation. The action of suppression on P. cinnamomi needs to be investigated further in trials within rehabilitated bauxite pits and forests, to fully understand the nature of the biological control, before any rehabilitation management strategies involving Acacia species can be instigated. Acknowledgments This research was supported by Alcoa World Alumina Australia. We thank Samantha Jarvis and Tony Passchier (Alcoa) for mine site support; Sarah Collins, Meredith Fairbanks, Daniel Hüberli and fellow students (Murdoch University) for field and laboratory assistance; Shelley McArthur, Janet Webster and Lin Wong (Department of Conservation and Land Management) for technical assistance; and Matthew Williams (Department of Conservation and Land Management) for assistance with statistical analysis. References Bailey AM, Coffey MD (1986) Characterisation of microorganisms involved in accelerated biodegradation of metalaxyl and metachlor in soil. Canadian Journal of Microbiology 32, 562–569. Broadbent P, Baker KF, Waterworth Y (1971) Bacteria and actinomycetes antagonistic to fungal root pathogens in Australian soils. Australian Journal of Biological Sciences 24, 925–944. Bunny FJ (1996) The biology ecology and taxonomy of Phytophthora citricola in native plant communities in Western Australia. PhD Thesis, Murdoch University, Western Australia. Burrows ND (1985) Reducing the abundance of Banksia grandis in the jarrah forest by the use of controlled fire. Australian Forestry 48, 63–70. Burrows ND (1987) Fire caused bole damage to jarrah (Eucalyptus marginata) and marri (Eucalyptus calophylla). Department of Conservation and Land Management, Research Paper 3, Western Australia. Coffey MD (1991) Strategies for integrated control of soilborne Phytophthora species. In ‘Phytophthora’. (Eds JA Lucas, RC Shattock, DS Shaw, LR Cooke) pp. 441–432. (Cambridge University Press: Cambridge, UK)

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Manuscript received 12 June 2003, accepted 16 January 2004

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