Increased susceptibility of Eucalyptus marginata to stem infection by Phytophthora cinnamomi resulting from root hypoxia

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1 Plant Pathology (1999) 48, Increased susceptibility of Eucalyptus marginata to stem infection by Phytophthora cinnamomi resulting from root hypoxia T. Burgess a, J. A. McComb a, I. Colquhoun b and G. E. StJ. Hardy a a School of Biological Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia; and b Alcoa of Australia Limited, PO Box 252, Applecross, Western Australia 6153, Australia Eucalyptus marginata growing on rehabilitated bauxite mines may be exposed to waterlogging (hypoxia) at the roots, as well as ponding around the stems at the soil surface. This paper examines whether these conditions may predispose stems of E. marginata to infection by Phytophthora cinnamomi. Plants of E. marginata clones resistant and susceptible to P. cinnamomi were grown in an aeroponics system that could be sealed to allow the manipulation of oxygen levels in the root zone to simulate waterlogging. Plants grown under normal oxygen conditions were compared with those whose root zone was exposed to hypoxia (2 mg O 2 L ¹1 ) before, during or after the stems were inoculated with zoospores of P. cinnamomi. Inoculation was achieved by constructing receptacles around the stems that could hold water and zoospores. Stomatal conductance increased in plants whose roots had been exposed to hypoxia. This effect lasted for at least 2 weeks after the resumption of normal oxygen conditions. P. cinnamomi entered and colonized ponded stems; however, there were no visible lesions on stems 14 days after inoculation. For any given clone of E. marginata, the extent of colonization was significantly greater in stems whose root zone had been exposed to hypoxia than in control stems. The activity of the plant defence-associated enzymes PAL, 4-CL and CAD and the concentrations of soluble phenolics were higher in the stems of plants whose roots were exposed to hypoxia, but the increase in activity in response to colonization was much greater for plants whose roots were under normal aerobic conditions. The greatest difference between colonized and noninoculated plants was observed at the colonization front. Peroxidase activity increased after tissues were colonized, rather than preceding the colonization as seen with the other enzymes. The stress induced by root hypoxia remained after roots were returned to normal oxygen conditions. Plants with root hypoxia showed greater stem colonization by P. cinnamomi and seemed less able to recognize the pathogen and switch on rapid defence responses. Keywords: Eucalyptus marginata, Phytophthora cinammomi, plant defence, predisposition and stress, root hypoxia, stem infection Introduction During bauxite mining in the lateritic hills km south-east of Perth, Western Australia, the topsoil (0 5 1 m deep) is removed and stored and the gravel layer (up to 6 m) that contains bauxite is mined, thus exposing the kaolinitic clays that extend to the bedrock. In rehabilitated areas, the soil profile is composed of the replaced Correspondence: Dr Giles Hardy, School of Biological Sciences, Murdoch University, Western Australia, 6150, Australia ( g-hardy@central.murdoch.edu.au). Accepted 10 June topsoil overlying a disturbed layer of low-grade bauxite (0 4 m deep) over the undisturbed clays and bedrock (Nichols et al., 1985). These rehabilitated areas are ripped to a depth of approximately 1 2 m to break up soil compaction and rip lines are formed along the contour to prevent soil erosion (Ward et al., 1996). During ripping there is some mixing of topsoil with the disturbed bauxite layer and sometimes the clay layer. Depending on the composition of the ripped areas, clay can collect in the rip lines resulting in surface ponding after rain. In addition, the kaolinitic clay layer (1 m or more below the surface) can impede water infiltration, resulting in transient subsurface ponding of water (Nichols et al., 1985; Kinal et al., 1993). It is believed 1999 BSPP 797

2 798 T. Burgess et al. that in these soil profiles, subsurface ponding leads to waterlogging, which has an impact on plant growth and disease development. Eucalyptus marginata (jarrah) is a dominant overstorey tree species in south-western Australia (Dell & Malajczuk, 1989). Phytophthora cinnamomi has had a major impact on the jarrah forest ecosystem, where it kills not only jarrah, but also up to 40% of understorey species (Shearer & Tippett, 1989). Over the last 15 years, lines of E. marginata resistant to P. cinnamomi have been selected through underbark inoculation using mycelia (Stukely & Crane, 1994). From these selections, clonal lines have been developed and tested by planting them on infected sites with additional inoculum placed around the roots in the form of pine plugs colonized with several P. cinnamomi isolates (McComb et al., 1994). Hardy et al. (1996) observed that, in contrast to the P. cinnamomi infection patterns seen in undisturbed jarrah forest, on rehabilitated bauxite mine sites P. cinnamomi formed lesions in the collars of 1- to 7- year-old jarrah. These plants were invariably found in the rip lines and it was hypothesized that invasion of the collar by P. cinnamomi was associated with ponding of water around the plants for hours or days following rain. These ponds were up to 15 cm in depth and lasted up to 5 days after rain (ponding was continuous in a wet winter) (Burgess et al., 1999a). The oxygen levels in these ponds remained close to saturation and did not become hypoxic even as the ponds drained (Burgess et al., 1999a). These conditions are optimal for growth, sporangia production and zoospore germination by P. cinnamomi (Davison & Tay, 1986). O Gara et al. (1996) demonstrated that zoospores of P. cinnamomi in ponds can enter, infect and form lesions through unwounded periderm tissue of E. marginata. In waterlogged (hypoxic) soils, a combination of altered host physiology and increased zoospore motility is thought to result in increased root infection by soilborne pathogens (Schoeneweiss, 1975; Zentmyer, 1980). Waterlogging leads to the development of tyloses in the xylem vessels of roots of E. marginata (Davison & Tay, 1985). Under controlled conditions, the number of xylem vessels blocked by tyloses increased with the duration of waterlogging, with half of the xylem vessels blocked after 13 days of waterlogging at 20 C (Davison & Tay, 1985). Many plants respond to waterlogging by closing their stomata, but jarrah seedlings continued to transpire (Davison & Tay, 1985). Thus, the rate at which the young jarrah wilted and died depended upon the duration of waterlogging and the transpiration rate. In a survey of ponds in young rehabilitated vegetation at the Huntly minesite in 1997, 30% of ponds were associated with subsurface water that had low oxygen levels (Burgess et al., 1999a). Oxygen levels in the subsurface depressions dropped to below 35% of that of air-saturated water within 2 days of a rain event under winter conditions (Burgess et al., 1999a). If there was no further replenishing rain, oxygen levels continued to drop. Thus, the potential exists for zoospores of P. cinnamomi in ponds to infect the collar of jarrah while the plant is under physiological stress due to waterlogging of the root zone. It is not known if these conditions decrease the ability of jarrah to contain the developing lesion. In the present study, glasshouse experiments were established using an aeroponics system (Burgess et al., 1998) to simulate waterlogging in the root zone, while the stem was ponded and inoculated with P. cinnamomi using the technique of O Gara et al. (1996). Stomatal conductance was used as a nondestructive measurement of plant stress due to water deficit and assays of plant defence enzymes in the stem were made to determine if root hypoxia reduced the ability of P. cinnamomi-resistant and P. cinnamomisusceptible jarrah clones to defend themselves from infection by P. cinnamomi. Materials and methods Plant material Clonal lines 1JN30 and 326J51 of E. marginata, previously ranked as resistant to P. cinnamomi, and line 11JN402, ranked as susceptible (Stukely & Crane, 1994), were used. Plants were grown in small pots (5 cm in diameter, 15 cm in length). When they were 5 months old (6 8 leaves), new root growth was encouraged by removing the bottom 10 cm of the pot and embedding the remaining pot and the exposed root ball into peat/ perlite (2 : 1 v/v) potting mix containing basal nutrients in drained polystyrene boxes. Plants were watered twice daily for 10 min. Side-shoots were removed regularly and, after 8 weeks, leaves were removed from the basal 15 cm of the stem. After 10 weeks the plants, which by then had leaves and a basal diameter of about 5 mm, were transferred to the aeroponics chambers as described by Burgess et al. (1998, 1999b). Experiments commenced 4 weeks later. Aeroponics chambers were housed in controlled-temperature glasshouses (20 27 C). Gas manipulation The chambers were sealed and the gas levels manipulated by controlling the nitrogen flow through gas flow meters. Oxygen levels within the chambers were measured using a dissolved oxygen meter (WTW, Weilheim, Germany) calibrated on water-saturated air (humidity within the chamber was 100%). The oxygen probes were left in situ for the duration of the hypoxic treatments. The chambers were first flushed with nitrogen to obtain the desired oxygen level. Oxygen levels of 25% of normal (approximately 2 mg O 2 L ¹1 at 20 C) were selected for these experiments. Normal oxygen conditions refer to 100% air saturation at any given temperature. These hypoxic conditions were maintained by a flow rate of approximately 150 ml N 2 min ¹1. The gas within the chambers was mixed by regular misting of the roots with water. The oxygen delivered by the watering system was not

3 Hypoxia and Phytophthora stem infection in Eucalyptus 799 sufficient to alter the readings on the dissolved oxygen meter that was suspended within the aeroponics chamber. Zoospore production The highly pathogenic P. cinnamomi isolate 94 48, from a diseased jarrah from a rehabilitated mine at Willowdale, Western Australia (Hüberli, 1995), was used in this study. Cultures were maintained on vegetable juice (V8) agar and zoospores produced by the method of O Gara et al. (1996). Plugs of fresh mycelium were transferred onto V8 agar overlaid with a sterile cheesecloth square. After 4 days, the cheesecloth was transferred to a 250-mL conical flask containing 150 ml of V8 broth, shaken overnight at 150 r.p.m. in lit conditions, rinsed thoroughly with mineral salts (5 mm KNO 3, 10mM Ca 2 (NO) 3 H 2 O, 4 mm MgSO 4 ) and shaken overnight in mineral salts to induce the formation of sporangia. The next day, the flasks were placed on a light box for 2 h, cold-shocked for 20 min and then left at 22 4 C until zoospores were released. Receptacle for ponding and inoculation of stem Two weeks prior to the commencement of the first hypoxic treatment, periderm formation was induced by gently rubbing the lower 10 cm of the stem with finegrade wet and dry sandpaper. Periderm formation occurred within 3 days. Five days before inoculation, a watertight receptacle (disposable plastic cup) was constructed around the base of each stem using the method of O Gara et al. (1996). Briefly, the cup was cut to approximately 4 cm in depth. A heated cork borer was used to melt a hole fractionally larger than the stem in the centre of the cup base. The cup was cut down one side and across the bottom to the centre hole. A strip of Parafilm was wrapped around the base of the stem just below a node and the cup was placed around the stem over the Parafilm. Insulation tape was used on the outside of the cup to rejoin the slit and silicone glass sealant (Selleys Chemical Company Pty. Ltd, Australia) was applied internally around the stem and along the slit to produce a watertight receptacle. The sealant took 1 day to cure. A ponding duration of 5 days was selected, as it represented the duration observed for a slowly draining pond during a period of average rainfall (Burgess et al., 1999a). Experimental design There were three separate experiments: expt 1 in September 1997 with clone 326 J51, expt 2 in November 1997 with clones 1JN30 and 11JN402, and expt 3 in February 1998 with clone 11JN402. The second experiment will be discussed in detail. There were 16 plants of clonal line 1JN30 and 10 plants of clonal line 11JN402 in each of five aeroponics chambers. Each experiment ran for 30 days and all stems were exposed Figure 1 Experimental design of glasshouse experiments indicating the duration of (A) normal oxygen conditions and (B) root-zone hypoxia and the timing of stem ponding and (P) harvest. All stems from all treatments were ponded between days 11 and 16. Stems were inoculated with zoospores of Phytophthora cinnamomi after 2 days of ponding. All plants were harvested at day 30. to 5 days of ponding, between day 11 and day 16 (Fig. 1). In addition, roots were treated in the following ways: (a) normal oxygen conditions for the whole 30 days, coded 0P0; (b) root hypoxia for 11 days before ponding, normal oxygen during and after ponding, coded 11P0; (c) root hypoxia for 2 days before, during and for 2 days after stem ponding, coded 2P2; (d) root hypoxia for 14 days after ponding, coded 0P14; and (e) root hypoxia during the entire 30 days, coded 11P14. Two days before the addition of P. cinnamomi inoculum (day 11), 30 ml of distilled water was put into each cup to test for leaks and to soften the periderm, thus mimicking the effects of ponding as observed in rehabilitated mine sites. On the day of inoculation (day 13), all the water was removed (a sample was taken for phenolic analysis) and replaced with fresh water. Zoospores (final concentration approximately 100 ml ¹1 ) and approximately 5 mg dry weight of P. cinnamomi mycelium were added to 75% of the cups. The rest of the cups were noninoculated controls. Three days later (day 16), the cups containing the inoculum and the Parafilm were removed. Stems were marked at the top of the Parafilm using liquid paper (Gillette Australia Pty. Ltd, Australia). This was termed the inoculation point.

4 800 T. Burgess et al. Plants were harvested 2 weeks after ponding finished (day 30). A 10-cm length of stem above the inoculation point was divided into 1-cm segments, which were then split longitudinally. One half of each segment was plated onto P 5 ARPH Phytophthora selective medium (Jeffers & Martin, 1986); the other half was immediately frozen in liquid nitrogen and stored at ¹80 C for enzyme extractions. The protocol for the other 2 experiments was the same, but with fewer hypoxic treatments. In expt 1, resistant clone 326J51 was subjected to all treatments except (b). In expt 3, susceptible clone 11JN402 was subjected to treatments (a) and (e) only. Samples were not harvested for enzyme analysis in these experiments. The data were analysed using STATISTICA (StatSoft, Tulsa, USA). Means were compared by analysis of variance and are presented with the standard error of the mean. Porometer readings Stomatal conductance was measured on the most recently expanded leaf on each plant using a Delta-T AP4 cycling Porometer (Delta-T Devices Ltd, Cambridge, UK). Readings were taken every 3 4 days, from just before the commencement of the first hypoxic treatment (day 0 on Fig. 1). All measurements were made within an hour in the early afternoon and the sequence in which plants were measured changed daily. Enzyme extraction and analysis Enzymes were extracted from each of the first five 1-cm segments above the inoculation point of colonized and noninoculated resistant plants of clonal line 1JN30 using a modification of the method used by Moerschbacher et al. (1988). Enzymes were not extracted from susceptible plants. Plants selected for analysis had P. cinnamomi present for 3 cm above the inoculation point (as determined from the selective agar plates). Between 5 and 9 stems were examined for each treatment. The tissue was powdered in a mortar and pestle with liquid nitrogen and 25% w/w quartz sand. To this was added 1 ml of extraction buffer (0 1 M sodium borate (ph 8 8) containing 1 mm ethylenediaminetetraecetic acid, 1 mm dithiothreitol, 1 mm ascorbate and 1 mm phenylmethylsulphonyl fluoride). The extract was centrifuged in a 1 5-mL Eppendorf tube at g for 10 min at 4 C and 20 L of the supernatant removed to determine the concentration of soluble phenolics. Polyvinylpolypyrolidine (10% w/v) was added to the supernatant, mixed and the extract re-centrifuged. The supernatant was transferred into an Eppendorf tube containing 200 Lof glycerol, mixed thoroughly and stored on ice before being used for enzyme and protein analysis. The protein content of the supernatant was determined using Bradfords Protein Determination Kit (Biorad, Hercules, CA. USA) and expressed in mg ¹1 ml by comparing absorbance at 595 nm with that of a bovine serum album standard curve. The concentration of soluble phenolics was determined using Folin and Ciocalteau s Phenol reagent (Sigma) and expressed as mol mg ¹1 protein by comparing absorbance at 725 nm with that of a 4-coumerate standard curve. Phenylalanine ammonia lyase (PAL; EC ) activity was determined according to the method of Cahill & McComb (1992). Enzyme extract (50 L) was mixed with 3 ml of 0 1 M sodium borate containing 30 M of L- ord-phenylalanine. Absorbance at 290 nm was read immediately and after a 60-min incubation at 30 C. The amount of transcinnimate produced (determined from the difference between the absorbance of the D- and L-phenylalanine) was determined from a transcinnimate standard curve and PAL activity expressed at pmol transcinnimate s ¹1 mg ¹1 protein. 4-Coumerate coenzyme A ligase (4-CL; EC ) activity was determined according to the method of Moerschbacher et al. (1988). Enzyme extract (50 L) was mixed with 700 L of 0 2 M phosphate buffer (ph 7 3) containing 20 mm MgCl 2, 2mM ATP, 4 mm DTT and 0 4 mm 4-coumerate, and incubated at 30 C for 1 min. The reaction was started by the addition of 100 L of 0 2 M phosphate buffer containing 2 mm CoA. Absorbance at 333 nm was read immediately and after a 10-min incubation at 30 C. 4-CL activity was calculated as the increase in absorbance over 10 min and expressed as pkat mg ¹1 protein using the 4-coumaryl : CoA extinction coefficient of cm 2 mol ¹1. Cinnamyl alcohol dehydrogenase (CAD; EC ) activity was determined according to the method of Moerschbacher et al. (1988). Enzyme extract (50 L) was mixed with 400 L of 0 2 M Tris-HCl (ph 9 25) containing 0 3 M NADP þ and incubated at 30 C for 1 min. The reaction was started by the addition of 50 L of0 2MTris-HCl containing 2 mm coniferyl alcohol. Absorbance at 400 nm was read immediately and after a 10-min incubation at 30 C. CAD activity was calculated as the increase in absorbance over 10 min and expressed as pkat mg ¹1 protein using the coniferyl aldehyde extinction coefficient of cm 2 mol ¹1. Peroxidase (PO; EC ) activity was determined by modifying the method of Mozzetti et al. (1995). Enzyme extract (50 L) was mixed with 1 95 ml of 50 mm phosphate-citrate buffer (ph 5 8) containing 20 mm of guaiacol. After 1 min at 30 C, the reaction was started by the addition of 50 L of 0 6 M H 2 O 2. After 6 min, absorbance at 470 nm was compared with a reaction blank and PO activity expressed as pkat mg ¹1 - protein using the tetraguaiacol extinction coefficient of cm 2 mol ¹1. Results Plant stress Stomatal conductance of the nonstressed control plants (0P0, Fig. 1) varied considerably between days, depending upon time of day, ambient temperature and

5 Hypoxia and Phytophthora stem infection in Eucalyptus 801 showed increased stomatal conductance after 2 days of root hypoxia for the resistant plants (Fig. 2a) and 6 days for the susceptible plants (Fig. 2b). Results with clonal line 326J51 were similar. In the absence of ponding, stomatal conductance did not differ significantly from the controls until after 11 days of root hypoxia. For the treatment exposed to hypoxia after ponding (0P14), stomatal conductance was significantly greater than the controls after 4 days of hypoxia (day 20). Figure 2 Stomatal conductance (as a percentage of the nonhypoxic control, 0P0) for treatments 11P0 (A), 2P2 (S), 0P14 (K) and 11P14 (W). (a) Phytophthora cinnamomi-resistant Eucalyptus marginata clone 1JN30 and (b) P. cinnamomi-susceptible E. marginata clone 11JN402. Filled symbols were significantly different (P < 0 05) from the nonhypoxic control. Colonization of the stem by P. cinnamomi The soluble phenolic content of water collected from the cups before inoculation was greater (P < 0 05) for plants of the resistant clone 1JN30 ( mol cm ¹1 stem) than for plants of the susceptible clone 11 J402 ( mol cm ¹1 stem). There were no visible lesions on stems 14 days after inoculation in any of the experiments. However, by plating out 1-cm segments of stem on selective agar, P. cinnamomi was found to have colonized the stem up to 10 cm above the point of inoculation. The extent of stem colonization was greater (P < 0 05) in plants of E. marginata clones selected for resistance to P. cinnamomi than in plants of the susceptible clone (Fig. 3). Non-inoculated plants were not colonized by P. cinnamomi. For any given clone of E. marginata, the extent of colonization was greater (P < 0 05) in stems whose root zone had been exposed to hypoxia than in control stems (Fig. 3). There was no significant difference in colonization between any of the hypoxic treatments (Fig. 3). light intensity. Thus, stomatal conductance of the treated plants is presented as a percentage difference (increase or decrease) compared with the 0P0 control plants (Fig. 2). Inoculation with P. cinnamomi had no significant effect on stomatal conductance and the data presented for each treatment are the means of all inoculated and noninoculated plants. Although, on average, plants of the susceptible E. marginata clone 11J402 had a lower stomatal conductance than those of the resistant clone 1JN30, plants of both clones followed similar patterns in response to the imposed treatments (Fig. 2). For the two treatments subjected to root hypoxia before ponding of the stem, 11P0 and 11P14, stomatal conductance increased significantly (P < 0 05) from the controls, 0P0, only after 11 days of root hypoxia. However, stomatal conductance remained higher until harvest (day 30), whether root hypoxia continued (11P14) or ceased (11P0) (Fig. 2). The short hypoxic treatment, 2P2, gave an increased stomatal conductance after only 2 days of root hypoxia. The treatment that was exposed to root hypoxia after ponding, 0P14, Figure 3 Colonization (cm) in stems of Eucalyptus marginata following ponding and inoculation of the stem with zoospores of Phytophthora cinnamomi. Experiment 1 in September 1997 with clone 326 J51 (p), expt 2 in November 1997 with clones 1JN30 (A) and 11JN402 (o) and expt 3 in February 1998 with clone 11JN402 (B). The root zone of the plants had been exposed to different hypoxic treatments (see Fig. 1). Clones 326J51 and 1JN30 are classified as resistant to P. cinnamomi, clone 11JN402 as susceptible. Bars denote SE.

6 802 T. Burgess et al. Host defence enzymes Enzymes were extracted from each of the first five 1-cm segments above the inoculation point, 14 days after the removal of the inoculum receptacle or cup. Under normal oxygen conditions (0P0, Fig. 1), the activity of enzymes from the phenylpropanoid pathway (PAL, 4-CL and CAD), the concentrations of soluble phenolics and peroxidase activity were no different between the attachment area and higher parts of the stem. If the attachment of the inoculum receptacle to the stem had produced a wound response, then enzyme activity would have been higher in the first segment. There was no significant difference in enzyme activity between the hypoxic treatments, and data from the different treatments were pooled for comparison with the control treatment. Root hypoxia significantly (P < 0 05) induced enzyme activity in noninoculated stems. Averaged over all 5 segments, the concentration of soluble phenolics was 40% higher, 4-CL and CAD activity 50% higher and peroxidase activity 100% higher in stems of plants whose roots had been exposed to root hypoxia; PAL activity was 20% lower (data not shown). PAL, 4-CL and CAD activity and the concentrations of soluble phenolics did not differ significantly between stem segments from noninoculated plants whose roots had been exposed to hypoxia (see Fig. 4a for 4-CL). Figure 4 Enzyme activity in colonized stems (B,X) of resistant Eucalyptus marginata clone 1JN30 compared with noninoculated stems (A,W). All plants selected for analysis had Phytophthora cinnamomi present for 3 cm above the inoculation point. Roots were grown under normal (B,A) oxygen conditions or subjected to root hypoxia (X,W) before inoculation of the stems with zoospores of P. cinnamomi. P. cinnamomi was isolated from the first 3 cm of all the inoculated stems used for enzyme analysis. Colonization increased (P < 0 05) the activity of all enzymes and the concentrations of soluble phenolics in the first 5 cm of stems of these plants, whether their roots were grown under normal oxygen conditions or exposed to root hypoxia (4-CL and peroxidase illustrated in Fig. 4). PAL, 4-CL and CAD activity and the concentrations of soluble phenolics were higher in hypoxia-treated plants (for example Fig. 4a), but the response to infection was greater (P < 0 05) in normal plants (Fig. 5a d). For plants grown under normal oxygen conditions, the greatest difference between colonized and noninoculated plants was observed at the colonization front, 4 cm above the inoculation point (Fig. 5a d). Peroxidase activity followed a different pattern to that of the other enzymes. It was higher (P < 0 05) in the first segment of noninoculated stems from the hypoxic treatment than in the noninoculated controls (Fig. 4b). Inoculation increased peroxidase activity in both normal and hypoxia-treated plants, with the increase being much greater in the stems of normal plants (Fig. 5e). Peroxidase activity was highest in the segments closest to the inoculation point. Peroxidase activity increased after tissues were colonized, rather than preceding colonization as seen with the other enzymes. Discussion P. cinnamomi entered and colonized ponded stems of clonal E. marginata classified as resistant or susceptible to P. cinnamomi. This result supports earlier findings (O Gara et al., 1996, 1997) which demonstrated that P. cinnamomi could enter and infect through axillary buds or scars or through unwounded lower stem periderm of E. marginata seedlings. In the current study, colonization extended further from the inoculation point in plants of the resistant clones. The clones had been classified on their ability to resist root infection (McComb et al., 1994) and by underbark inoculation using mycelium (Stukely & Crane, 1994) and may not have equivalent capabilities when confronted with zoospore inoculation of the stem. At the time of harvest (14 days after inoculation), lesions had not yet developed in any plants; it is possible that, if left longer, the resistant clones may have been able to contain the lesions sooner than the susceptible clones. Flooded soils are assumed to increase the severity of diseases caused by root pathogens, primarily by adversely affecting host physiology while increasing the mobility of the pathogen through the soil (Schoeneweiss, 1975; Zentmyer, 1980). Davison & Tay (1985) demonstrated that during waterlogging (oxygen levels of less than 1 mg O 2 L ¹1 within one day of inundation), jarrah seedlings developed tyloses in the xylem of roots, which blocked the movement of water flow to the tops. The longer the duration of waterlogging, the more tyloses developed. They also found that in jarrah the stomata remained open until the leaves wilted and died.

7 Hypoxia and Phytophthora stem infection in Eucalyptus 803 Thus, the plants recovered after being waterlogged for 3 days, but died after longer durations. Tyloses were not examined in the current experiment. However, no plants wilted and died even after 14 days of root hypoxia (oxygen level maintained at approximately 2 mg O 2 L ¹1, or 25% of normal). The waterlogging/anoxic conditions used by Davison & Tay (1985) in glasshouse experiments produced lower oxygen levels (< 10%) than those observed in saturated soils in rehabilitated bauxite mines (30 35%, Burgess et al., 1999a) or in subsurface flows in mature jarrah forests (approximately 50%, Davison & Tay, 1991). Anoxic conditions obviously have a more adverse effect on E. marginata then the hypoxic conditions used in this experiment. Young jarrah experiencing water stress may still continue to transpire and give low stomatal conductance (Davison & Tay, 1985). However, when an increase in stomatal conductance is recorded, the plants are most likely experiencing stress due to water deficit. In the current experiments, measurements of stomatal conductance indicated that plants did not exhibit stress due to water deficit in the leaves unless exposed to more than 11 days of root hypoxia. However, plants that were ponded at the same time as root hypoxia was imposed exhibited stress almost immediately, and plants that were exposed to root hypoxia after ponding exhibited stress within 6 days of the commencement of root hypoxia. This would suggest that ponding contributed to plant stress; however, as plants under normal conditions did not become stressed by ponding alone, it appears that ponding is only effective in conjunction with root hypoxia. In these experiments, all plants, whether inoculated or not, were ponded. Thus, the effects of ponding and root hypoxia on plant stress cannot be separated. There is little information on the effect of waterlogging on above-ground disease development, but it can be assumed that stressed plants are less able to defend themselves from pathogen invasion. In the current study, exposure of the E. marginata root zone to simulated waterlogging (root hypoxia) increased the colonization of P. cinnamomi in jarrah stems. All the hypoxic treatments examined had an equally stimulatory effect on colonization. Neither the duration of root hypoxia nor its timing in relation to the ponding of stems affected the final extent of P. cinnamomi stem colonization. When root hypoxia was imposed before inoculation, the plants were stressed and the pathogen entered the stem rapidly; when root hypoxia was imposed after ponding, then P. cinnamomi present on the stem (but not growing Figure 5 The percentage increase in enzyme activity in colonized stems of resistant Eucalyptus marginata clone 1JN30 compared with noninoculated stems. All plants selected for analysis had Phytophthora cinnamomi present for 3 cm above the inoculation point. Roots were grown under normal (A) oxygen conditions or subjected to root hypoxia (B) before inoculation of the stems with zoospores of P. cinnamomi.

8 804 T. Burgess et al. rapidly) could take advantage of the induced stress and penetrate rapidly. Leaving the plants longer before harvesting may have given different results, with the treatments having continued root hypoxia after ponding expected to have more rapid stem colonization. However, the readings of stomatal conductance indicated that plants did not recover from hypoxia-induced stress once normal oxygen treatments were re-established, and thus stem colonization may also have been rapid in these plants. P. cinnamomi is a necrotroph that forms lesions in diseased tissue (Zentmyer, 1980). Its success as a pathogen depends upon its ability to enter host tissue and overcome host defence responses. Complete resistance to necrotrophic diseases is rare, but some plants are more able than others to contain and overcome infection and these are selected for breeding programmes; this is how the E. marginata clones used in these experiments were selected (McComb et al., 1994). Plants have evolved a complex array of defence mechanisms including the accumulation of toxic phenols, modification of cell walls by phenolic substitution or physical barriers such as lignin and suberin, and the synthesis of phytoalexins (Hahlbrock & Scheel, 1989; Bowles, 1990; Nicholson & Hammerschmidt, 1992; Kuc, 1995). All these mechanisms are by-products of the phenylpropanoid pathway, and the induction of enzymes from this pathway (such as PAL, 4-CL and CAD) and the accumulation of its products are indicators of active plant defence. Cahill & McComb (1992) demonstrated that PAL activity and lignin and phenolics accumulation increased after root infection of field-resistant Corymbia calophylla (formerly E. calophylla) seedlings and plants of a resistant E. marginata clone with P. cinnamomi. These changes occurred to a much lesser extent in roots of susceptible plants (Cahill et al., 1993). Although susceptible plants had the mechanisms required for resistance, they were unable to activate these defence reactions in time or with sufficient intensity to effectively restrict pathogen invasion. When looking at plant defence enzymes in response to a pathogen under conditions of stress (in this case root hypoxia), the responses are more complicated, as the abiotic stress also induces plant defence responses (Ayres, 1984; Dixon & Paiva, 1995). In the current study, enzyme induction due to root hypoxia and P. cinnamomi inoculation was examined for a single experiment and there is a possibility that repetition of this experiment could produce different results. However, the results follow logical patterns and warrant discussion here. Root hypoxia induced the activity of enzymes from the phenylpropanoid pathway and their products in the stems of affected plants. Colonization by P. cinnamomi further increased the activity of all these enzymes. Actual levels of enzyme activity were higher in plants that had been exposed to hypoxia, but the relative increase in enzyme activity in response to the pathogen was much less than in control plants grown under normal conditions. It appears that, in this study, the rapid increase in enzyme activity was more important than the actual level of enzyme activity in slowing colonization by P. cinnamomi. Perhaps during hypoxia the products of these enzymes had a more general function than plant defence. Alternatively, in control plants the increased enzyme activity could have been targeted to the site of pathogen invasion while in the stressed plants there was no specificity in the localization of enzyme activity. Thus, the containment of colonization by P. cinnamomi involved the temporal and spatial induction of plant defence enzymes from the phenylpropanoid pathway. This is particularly important as P. cinnamomi was moving up the stems without producing visible lesions. O Gara et al. (1997) had noticed this phenomenon in the field and considered that P. cinnamomi could act as a hemi-biotroph in some situations. Plants need to be left longer than 14 days after inoculation to determine whether disease development leads to death in plants stressed by root hypoxia, while being arrested in control plants. Peroxidases are involved in many cell processes and their presence has been demonstrated in various subcellular components (Parish, 1975; Fric, 1976; Lee & Lin, 1995). Changes in peroxidase activity and isozyme composition are not a specific reaction to infection, but rather an accompanying characteristic of changed metabolic activity of a plant cell under stress (Fric, 1976). Thus, peroxidase induction is not necessarily indicative of a plant defence response. Burgess et al. (1997) found that soluble peroxidase activity increased in the apical regions of roots during hypoxia. In the current study, peroxidase activity was high in noninoculated but stressed plants (those whose roots were exposed to hypoxia). Soluble peroxidase activity was also high in colonized tissue, but not beyond the colonization front. In these plants peroxidase induction appears to reflect tissue damage rather than plant defence. In conclusion, root hypoxia (simulated waterlogging) caused stress to plants that remained after roots were returned to normal conditions. This stress, in association with P. cinnamomi infection introduced by stem ponding, led to greater disease development than that experienced by plants whose roots had not been exposed to hypoxia. The stressed plants seemed less able to switch on rapid defence responses against the pathogen. Acknowledgements This project was funded by an Industry Collaborative Grant from the Australian Research Council. References Ayres PG, The interaction between environmental stress injury and biotic disease physiology. 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