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Role of hydrogen peroxide during the interaction between the hemibiotrophic fungal pathogen Septoria tritici and wheat Blackwell Publishing Ltd
Nandini P. Shetty1, Rahim Mehrabi2, Henrik Lütken1, Anna Haldrup1, Gert H. J. Kema2, David B. Collinge1 and Hans Jørgen Lyngs Jørgensen1 1
University of Copenhagen, Faculty of Life Sciences, Department of Plant Biology, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark;
2
Plant Research International, Wageningen University and Research Centre, PO Box 16, 6700 AA Wageningen, the Netherlands
Summary Author for correspondence: N. P. Shetty Tel: +45 35 28 23 23 Fax: +45 35 28 33 10 Email:
[email protected] Received: 11 September 2006 Accepted: 5 January 2007
• Hydrogen peroxide (H2O2) is reported to inhibit biotrophic but benefit necrotrophic pathogens. Infection by necrotrophs can result in a massive accumulation of H2O2 in hosts. Little is known of how pathogens with both growth types are affected (hemibiotrophs). The hemibiotroph, Septoria tritici, infecting wheat (Triticum aestivum) is inhibited by H2O2 during the biotrophic phase, but a large H2O2 accumulation occurs in the host during reproduction. • Here, we infiltrated catalase, H2O2 or water into wheat during the biotrophic or the necrotrophic phase of S. tritici and studied the effect of infection on host physiology to get an understanding of the survival strategy of the pathogen. • H2O2 removal by catalase at both early and late stages made plants more susceptible, whereas H2O2 made them more resistant. H2O2 is harmful to S. tritici throughout its life cycle, but it can be tolerated. • The late accumulation of H2O2 is unlikely to result from down-regulation of photosynthesis, but probably originates from damage to the peroxisomes during the general tissue collapse, which is accompanied by release of soluble sugars in a susceptible cultivar. Key words: hemibiotrophic, hydrogen peroxide (H2O2), Mycosphaerella graminicola, Septoria tritici, stress, wheat (Triticum aestivum). New Phytologist (2007) 174: 637 –647 © The Authors (2007). Journal compilation © New Phytologist (2007) doi: 10.1111/j.1469-8137.2007.02026.x
Introduction One of the earliest of many diverse defence reactions activated in plant tissues in response to pathogen attack is the accumulation of reactive (or active) oxygen species (ROS) (Bolwell et al., 1995; Bolwell, 1999; Apel & Hirt, 2004), which include the superoxide radical (O2–), hydrogen peroxide (H2O2), and the hydroxyl radical (OH•). The phenomenon of ROS accumulation is often termed the oxidative burst. Chemically, H2O2 is the most stable of the ROS species, and hence the most easy to study (Costet et al., 2002). H2O2 has a direct antimicrobial effect and is involved in cross-linking in cell walls, induction of gene expression, hypersensitive cell death,
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phytoalexin production and induced systemic resistance (Peng & Kuc, 1992; Wu et al., 1995; Thordal-Christensen et al., 1997; Bolwell, 1999; Costet et al., 2002; Apel & Hirt, 2004; Gechev & Hille, 2005). Several studies have attempted to elucidate the role of H2O2 in host–pathogen systems. For example, it has been reported that biotrophic pathogens are inhibited by H2O2 accumulation (Thordal-Christensen et al., 1997; Vanacker et al., 2000; Mellersh et al., 2002), whereas necrotrophic pathogens are reported to be favoured by H2O2 production or even to stimulate its production (Gönner & Schlösser, 1993; Govrin & Levine, 2000; Kumar et al., 2001; Able, 2003). The question is, then, as follows: how do pathogens exhibiting characteristics of both lifestyles, that is, the so-called
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hemibiotrophic pathogens, react to H2O2 during their different phases of growth? In order to understand the role of ROS in relation to pathogen biology and to avoid oversimplifications about this role, it is necessary to study several different host– pathogen interactions, involving monocots and dicots as well as different types of pathogens. Subsequently, this information will provide the basis for advanced molecular and genetic studies to verify the mechanisms of formation and role of individual ROS beyond any doubt. The interaction between wheat (Triticum aestivum) and Septoria tritici, the causal agent of speckled leaf blotch or septoria tritici blotch, is currently being established as a model system for studies involving hemibiotrophic pathogens. Defence response studies (Ray et al., 2003; Shetty et al., 2003; N. P. Shetty, unpublished) and full genome sequencing of the pathogen have been initiated (http://www.jgi.doe.gov/sequencing/ why/CSP2005/mycosphaerella.html). Recently, Shetty et al. (2003) found that the infection of wheat by S. tritici was associated with a large and early accumulation of H2O2 in incompatible interactions, coinciding with pathogen arrest and thus indicating a role for H2O2 in the active defence of wheat. In a compatible interaction, very little H2O2 accumulated during the initial biotrophic phase of the interaction, where hyphae grew in the host apoplast after initial stomatal penetrations. This phase lasted until c. 11 d after inoculation (dai) and was followed by a massive accumulation of H2O2, tissue collapse and necrosis, coinciding with pathogen sporulation (c. 5 dai). This pattern suggests that H2O2 production is a defence response during the early biotrophic phase of the interaction, whereas the pathogen thrives with the late, massive accumulation of H2O2 during the necrotrophic phase. In this study, we manipulate H2O2 concentrations in wheat to determine the causal relationship between H2O2 and the amount of fungal infection in the interaction with S. tritici. This was achieved by scavenging H2O2 with catalase or supplementing with additional H2O2. Furthermore, we attempted to elucidate the origin of the late accumulation of H2O2 by measuring the amounts of chlorophyll and soluble sugars in the leaves to determine the effect of the pathogen on photosynthesis. Based on these data, we propose a hypothesis to explain the adaptation of lifestyle adopted by S. tritici to survive and reproduce in wheat.
Materials and Methods Plants and inoculation Isolate IPO323 of Septoria tritici Roberge in Desmaz. (teleomorph: Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn) was used as inoculum, and applied to 14-d-old plants of wheat (Triticum aestivum L.) of cv. Sevin (susceptible) and cv. Stakado (resistant), as described previously (Shetty et al., 2003).
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Infiltration of catalase, H2O2 and water Leaves were infiltrated with catalase (2000 U ml−1, C-9322, Sigma, St Louis, MO, USA), H2O2 (4 mM, H-1009, Sigma) or distilled water (control treatment), using a plastic syringe without a needle, until completely saturated. To investigate the role of H2O2 during the biotrophic phase of the interaction, infiltration took place immediately before inoculation with S. tritici and again 3 and 5 dai. Leaves were harvested 9 dai and cleared as described by Shetty et al. (2003). For each time point and treatment, four leaves were harvested. After clearing, fungal development on each leaf was studied by light microscopy. For 100 spores on each leaf, the numbers that produced (i) germ tubes reaching a stoma, (ii) appressoriumlike swellings, and (iii) penetration were recorded. The extent of hyphal colonization after penetration was recorded in a qualitative manner and symptom development was scored visually until 21 dai on a daily basis. In order to study the effect of H2O2 during the necrotrophic phase of the interaction, leaves were infiltrated with catalase, H2O2 or water (control) at 9, 11 and 13 dai. Four leaves for each treatment and time point were harvested 15 dai and cleared as before. After clearing, the extent of fungal colonization was examined for 500 stomata on each leaf. Fungal growth was divided into three categories; (i) the presence of individual hyphae in the substomatal cavities; (ii) the presence of pycnidial initials; and (iii) the presence of fully developed, sporulating pycnidia. To test whether the effect of catalase or H2O2 observed after infiltration could also be obtained by external application, leaves were sprayed until run-off with either catalase, H2O2 (same concentrations as before) or water, and the effect on fungal development determined. Leaves were sprayed either early (before inoculation, 3 and 5 dai) or late (9, 11, 13 dai). Harvest of leaves and their examination took place as in the corresponding infiltration experiments. As a second type of control, leaves were infiltrated with catalase (2000 U ml−1) which had been boiled for 5 min and symptom expression was compared with that of leaves infiltrated with catalase or water as before. Quantification of fungal DNA Twenty leaves from experiments with early infiltration of catalase, H2O2 or water were prepared as described above and immediately frozen in liquid nitrogen after harvest. The leaves were subsequently freeze-dried over night and ground using a bead beater (Hybaid Ribolyser, model FP120HY-230, Thermo Electron Corporation, Delft, the Netherlands) for 20 s at 6500 r.p.m. with a tungsten carbide bead until the leaves were totally macerated. About 5 mg of the fine leaf powder was transferred to new tubes for DNA isolation, which was performed using the DNeasy® plant mini kit (Qiagen, Venlo, the Netherlands). Two microlitres of diluted DNA were used for quantitative PCR using TaqMan technology, with primers
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and probe designated for M. graminicola mating type gene 1-1 (GenBank accession number AF440399) (Waalwijk et al., 2002; Ben M’Barak et al., 2003). Quantitative PCR amplification of 2 µl of template was performed in a 30 µl total volume reaction mixture containing 0.75 U Hot GoldStar™ polymerase, 1X reaction buffer, 5 mM MgCl2, 200 µM of each dNTPs, 0.3 µM of each primer, all reagents from Eurogentec (Maastricht, the Netherlands), and a 0.08 µM probe. PCR amplification and quantitative analysis were performed in an Abi Prism 7700 sequence detector (Applied Biosystems, Foster City, CA, USA). The PCR reaction profile consisted of an initial period (2 min at 50°C) to degrade uracil containing DNA, a Hot GoldStar polymerase activation interval (10 min at 95°C), followed by 40 cycles of repeated denaturation (15 s at 95°C) and annealing/ extension (60 s at 65°C). The DNA was quantified based on the serial dilution (0, 2, 20, 200, 2000 and 20 000 pg) of known DNA of S. tritici (isolate IPO323), using the Abi Prism 7700 Sequence Detection software system. To detect samples containing potential PCR inhibitors, 20 pg of an internal positive control (Potato leaf roll virus) was added into each PCR reaction and amplified separately using specific primers and probes, as described previously (Waalwijk et al., 2004). A negative result for the target and a positive result for the internal control DNA indicate absence of inhibitors.
UK). The chlorophyll fluorescence transients were induced by red light of λ = 650 nm with an intensity of 3000 µmol photons m−2 s−1 and recorded during 2 s. All measurements were made on dark-adapted leaves (30 min adaptation). The ((Fm − F0 )Fm−1) = Fv /Fm, maximum quantum efficiency of PSII, was determined (Christensen et al., 2003). At each time point and treatment, 12 independent recordings were made. Estimation of total chlorophyll content After inoculation or treatment with water, six leaves were collected from each time point and incubated at 80°C for 1 h in 1 ml 96% (v/v) ethanol until the leaves were completely devoid of pigments. Subsequently, the absorbance at λ = 648.6 nm and λ = 664.2 nm was measured using a spectrophotometer and the ratio between chlorophyll a and b estimated (Lichtenthaler, 1987). Measurements were made in triplicate on each of the 10 replicates per combination of cultivar and treatment. Extraction of soluble sugars
In vitro experiments were carried out to study the inhibitory effect of varying concentrations of H2O2 on the growth of the pathogen. S. tritici inoculum was produced as before (Shetty et al., 2003) and harvested from either 4- or 16-d-old cultures. Different concentrations of H2O2 were prepared (50, 100 and 500 mM) in distilled water and mixed well with a suspension (1 : 9 ratio, v/v) of S. tritici containing 106 spores ml−1 in Eppendorf tubes, thus giving final H2O2 concentrations of 5, 10 and 50 mM. Controls consisted of water and a catalase treatment where the spore suspension was mixed with a catalase solution (2000 U ml−1) in a 1 : 9 ratio (v/v). All treatments were repeated three times. Following pilot experiments using time intervals from 60 to 600 s, a 180 s incubation period was chosen since it gave a very clear and differentiating effect. Subsequently, 30 µl from each tube were spread on 5 cm PDA plates that were incubated for 6 d as for inoculum production (Shetty et al., 2003). The number of colonies on each dish was counted, using a light microscope (10 random fields at ×10 magnification, total area in field of vision approx. 2.4 mm2).
After inoculation or treatment with water, leaves were harvested and sugars (glucose, fructose and sucrose) extracted essentially as described by Müller-Röber et al. (1992). Leaf segments (approx. 100 mg) were incubated at 80°C for 1 h in 1 ml 80% (v/v) ethanol. The supernatant was used for enzymatic sugar determination according to Nielsen et al. (1991). The contents were measured in microtitre plates using a spectrophotometer. Thirty microlitres of plant extract were mixed with 200 µl assay buffer (50 mM MOPS/KOH, pH 7.3, 5 mM MgCl2 and 1 mM EDTA). Then 2.5 µl of 100 mM adenosine triphosphate (ATP), 5 µl of 50 mM NAD and 2.5 µl of 400 U ml−1 hexokinase were added. The absorbance at 340 nm was monitored until stable and 1 µl of 500 U ml−1 glucose-6-phosphate dehydrogenase was added. The absorbance at 340 nm was further followed until stable and 1 µl of 500 U ml−1 phosphoglucoseisomerase was added. Again the absorbance at 340 nm was followed until stable and 1 µl of 20 U µl−1 invertase was added. The concentration of the sugars was determined as the difference in absorbance after adding glucose-6-phosphate dehydrogenase, phosphoglucose isomerase and invertase for glucose, fructose and sucrose, respectively, and calculated from a standard curve generated from different amounts of 1 µl glucose, fructose and sucrose per µl, respectively. All the enzymes used were obtained from Sigma. Double measurements were made on all of the three biological replications per combination of cultivar and treatment.
Measurement of photosynthesis
Statistical analysis
Chlorophyll a fluorescence was measured daily (from 3 to 15 dai) in cvs Sevin and Stakado, for both plants inoculated with S. tritici or treated with water, using a portable plant efficiency analyser (Handy PEA, Hansatech Instruments Ltd, Norfolk,
Data from all microscopy studies represent discrete variables and hence were analysed by logistic regression, assuming a binomial distribution, as described by Shetty et al. (2003). Colony counts for assessing the effect of different concentrations
Effect of H2O2 on in vitro growth of S. tritici
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Fig. 1 Fungal growth and symptoms of Septoria tritici at 13 d after inoculation (dai) following early infiltration (0, 3 and 5 dai) of catalase, hydrogen peroxide (H2O2) or water. (a–c) Macroscopic symptoms of S. tritici infection in wheat (Triticum aestivum) cv. Sevin after infiltration of catalase (a), H2O2 (b) or water (c). (a) Leaves are almost completely wilted; (b) no symptoms of infection; (c) chlorotic and necrotic symptoms have started to appear. (d–i) Whole cleared leaves of cvs Sevin (d–f) and Stakado (g–i), stained with Evans blue after infiltration of catalase (d, g), H2O2 (e, h) or water (f, i). (d) Note pycniospores (Ps) from fully developed pycnidium (Py); (e, h) leaves infiltrated with H 2O2; (f, i) leaves infiltrated with water; (e, f, i) note varying numbers of internal hyphae (Hy) in and around stomata (arrows). (f) A pycnidial initial (Pi) is seen, whereas superficial hyphae (Shy), but no internal hyphae, are noted in (h).
of H2O2 on growth of S. tritici were also analysed by logistic regression, assuming a Poisson distribution. Here, the original data were subjected to statistical analysis, but results are presented as relative values, where the value in the control treatment (0 mM H2O2) was set to 100%. Data from measurement of photosynthesis represent a continuous variable and were analysed by analysis of variance assuming a normal distribution. Variances were stabilized by the appropriate transformation of data, if necessary. In the following, all differences are significant at P ≤ 0.05 unless specifically mentioned.
Results Infiltration of catalase, H2O2 and water Early infiltrations (0, 3 and 5 dai) in cv. Sevin resulted in a clear change in symptom expression. Thus, symptoms appeared
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earlier after infiltration of catalase and were delayed after infiltration of H2O2 compared with infiltration of water (Fig. 1a–c), and sporulation occurred 11, 21 and 15 dai after infiltration of catalase, H2O2 or water, respectively (data not shown). The infiltration of these substances alone (without inoculation) did not have any visible effect on the plants, and, furthermore, infiltration of water did not cause measurable changes in fungal behaviour compared with uninfiltrated leaves (data not shown). Microscopy of leaves after early infiltration was performed 9 dai in both cvs Stakado and Sevin (Table 1). In both cultivars, infiltration with catalase resulted in higher numbers of spores with germ tubes ending on stomata, producing appressorium-like swellings on stomata and penetrating through stomata compared with the control treatment. The penetration efficiency of the appressoriumlike swellings was higher in catalase-infiltrated leaves for cv. Sevin. Infiltration with H2O2 reduced all parameters examined
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Research Table 1 Percentage of Septoria tritici spores causing infection in leaves of wheat (Triticum aestivum) of the susceptible cv. Sevin and the resistant cv. Stakado at 9 d after inoculation (dai) following early infiltration of catalase, hydrogen peroxide (H2O2) or water (0, 3 and 5 dai) Treatment
cv. Sevin Spores with germ tubes ending on stomata Spores with swellings on stomata Spores causing penetration Swellings causing penetration cv. Stakado Spores with germ tubes ending on stomata Spores with swellings on stomata Spores causing penetration Swellings causing penetration
Odds ratio
Catalase
H2O2
Water
Catalase
H2O2
Water
26.5 25.3 23.3 91.3
11.8 10.3 6.0 60.6
13.3 10.5 6.8 64.5
2.38*** 2.93*** 4.28*** 6.66***
0.87 NS 0.97 NS 0.88 NS 0.81 NS
1.00 1.00 1.00 1.00
33.3 32.8 27.5 84.2
5.5 4.0 2.3 60.0
11.8 11.3 8.5 76.9
3.78*** 3.90*** 4.14*** 1.72 NS
0.41** 0.33*** 0.25*** 0.42 NS
1.00 1.00 1.00 1.00
Odds ratio for comparison of treatments (water used as a reference, odds ratio = 1.00). NS, nonsignificant difference; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.
Table 2 Percentage of stomata in cvs Sevin and Stakado having hyphal growth, pycnidial initials or fully developed pycnidia of Septoria tritici at 15 d after inoculation (dai) following late infiltration of catalase, hydrogen peroxide (H2O2) or water (9, 11 and 13 dai) Treatment
cv. Sevin Hyphal growth Pycnidial initials Fully developed pycnidia cv. Stakado Hyphal growth Pycnidial initials Fully developed pycnidia
Odds ratio
Catalase
H2O2
Water
Catalase
H2O2
Water
30.6 4.1 3.8
9.0 1.0 0.1
17.7 0.1 0.1
2.09 NS 43.83* 81.73***
0.45 NS 9.63 NS 1.00 NS
1.00 1.00 1.00
3.2 0.0 0.0
1.5 0.0 0.0
1.8 0.0 0.0
1.78 NS 1.00 NS 1.00 NS
0.80 NS 1.00 NS 1.00 NS
1.00 1.00 1.00
Odds ratio for comparison of treatments (water treatment used as a reference, odds ratio = 1.00). NS, nonsignificant difference; ***, P ≤ 0.001; *, P ≤ 0.05.
in cv. Stakado, except for the proportion of appressorium-like swellings leading to penetrations, compared with infiltration of water. In cv. Sevin, no significant differences were observed for the parameters examined. Hyphal growth after penetration increased considerably in cv. Sevin after treatment with catalase compared with water, the hyphae being more numerous and spread further following catalase treatment (Fig. 1d,f). In cv. Stakado, increased fungal proliferation was observed soon after penetration, but most hyphae did not grow past the substomatal cavities (Fig. 1g,i). In cv. Sevin, hyphal growth after treatment with H2O2 was sparse, with fewer and shorter hyphae compared with the water-treated control (Fig. 1e,f). The pathogen started to grow from the stomata into the substomatal cavities from 11 dai. Infiltration of H2O2 in cv. Stakado also reduced hyphal growth after penetration compared with the water treatment (Fig. 1h,i), with most hyphae stopping in the substomatal cavities, immediately after penetration.
Late infiltrations relating to the necrotrophic phase of the interaction took place 9, 11 and 13 dai, and data for 15 dai are presented in Table 2. Catalase generally resulted in enhanced fungal development. In cv. Sevin, the number of stomata with individual hyphae was higher, although not significantly, after infiltration of catalase compared with infiltration of water. On the other hand, a higher proportion of stomata had pycnidial initials and fully developed pycnidia after treatment with catalase than following water treatment. Infiltration of H2O2 did not significantly alter the extent of fungal growth compared with the water treatment. However, fewer stomata with individual hyphae were recorded, compared with catalase treatment (odds ratio = 0.22, P = 0.0269). In cv. Stakado, catalase and H2O2 infiltration did not significantly alter fungal colonization compared with the water treatment. Nevertheless, H2O2 infiltration resulted in a lower number of stomata with individual hyphae compared with the catalase treatment (odds ratio = 0.45, P = 0.0289). Pycnidial initials and fully
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Fig. 2 Quantification of Septoria tritici biomass in leaves of wheat (Triticum aestivum) cvs Sevin and Stakado by measuring the amount of fungal DNA by TaqMan. Each value is presented ± SE.
developed pycnidia were not seen after either treatment. Different control treatments were carried out for the infiltration experiments. Thus, some leaves were sprayed with catalase, H2O2 or water and others were infiltrated with boiled catalase to eliminate all enzyme activity. Fungal growth and symptom expression after these control treatments were not significantly altered compared with the infiltration of water (data not shown).
Fig. 3 Relative number of Septoria tritici colonies on potato dextrose agar after incubation of inoculum of different ages (4-d-old cultures, closed columns; 16-d-old cultures, open columns) in different concentrations of hydrogen peroxide (H2O2; 5, 10, 50 mM) or catalase (200 U) for 180 s. Control (0 mM H2O2) is set to 100%. Comparisons are possible within each inoculum age. NS, nonsignificant difference; ***, P ≤ 0.001; *, P ≤ 0.05.
Quantification of fungal DNA The fungal biomass after infiltration of H2O2, catalase or water was quantified by determining the amount of fungal DNA using TaqMan (Fig. 2). In cv. Sevin, there were no differences between any treatment at 3, 6 and 9 dai (P > 0.05), but for 12 and 15 dai, catalase infiltration resulted in higher fungal biomass than the two other treatments, which did not differ from each other (P = 0.0066, LSD = 1.6 and P < 0.0001, LSD = 2.0, respectively). No differences between treatments were observed in cv. Stakado (P > 0.05). Effect of H2O2 on in vitro growth of S. tritici In order to obtain an indication as to how much H2O2 S. tritici can tolerate, we performed in vitro experiments with different concentrations of H2O2. Two ages of inoculum were used, that is, harvested from 4- or 16-d-old cultures (Fig. 3). For the 4-d-old inoculum, a concentration of 5 mM H2O2 and the control with catalase did not significantly affect the survival of S. tritici after 180 s compared with the water control. Increasing the H2O2 concentration resulted in increasing inhibition of the pathogen. For the 16-d-old inoculum, none
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Fig. 4 Mean chlorophyll a fluorescence as an indirect measure of photosynthesis and mean chla + b content in wheat (Triticum aestivum) cvs Sevin and Stakado either inoculated with Septoria tritici or treated with water. Values are means ± SE.
of the concentrations of H2O2 tested significantly inhibited survival of S. tritici: only the catalase treatment resulted in a slight inhibition. Measurement of photosynthesis We examined whether the H2O2 accumulation occurring during the necrotrophic phase of the interaction could result
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from degenerating chloroplasts, which would be indicated by a reduction in photosynthesis. The influence of fungal infection and colonization on photosynthesis was studied indirectly by measuring the chlorophyll a fluorescence at 3–15 dai (Fig. 4). The values for all four combinations of cultivar (Sevin or Stakado) and pretreatment (inoculation with S. tritici or treatment with water) were all relatively stable around Fv/Fm = 0.80–0.83 during the entire experiment, with only minor fluctuations over time. However, for cv. Sevin inoculated with S. tritici, Fv/Fm showed a declining trend from 11 dai, followed by a sharp drop to 0.22 at 15 dai. Macroscopically, all leaves remained green without visible symptoms of infection or stress until 11 dai, when leaves of cv. Sevin developed small, necrotic spots, and at 15 dai, the leaves wilted completely and pycnidia were visible throughout the necrotic tissue. Determination of total chlorophyll content To test whether fungal infection resulted in reduced amounts or degradation of the chlorophyll in spite of the green appearance of the leaves, the total chlorophyll content was measured (Fig. 4). Generally, higher amounts of chlorophyll were extracted from cv. Sevin than from cv. Stakado. The values fluctuated between 0.8 and 2.5 µg mg−1 FW of the leaves, but were relatively stable over time for the individual treatments. Generally, inoculation resulted in a lower yield of chlorophyll than for the controls for both cultivars, but the differences were not significant. However, the chlorophyll content declined throughout the experiment in cv. Stakado inoculated with S. tritici, with lower values from 11 dai, and in inoculated cv. Sevin, which showed a higher chlorophyll content at 7 and again at 15 dai, compared with the other treatments. Extraction of soluble sugars To examine whether increased release of carbohydrates could explain repression of photosynthesis in cv. Sevin after inoculation with S. tritici, we measured the amounts of the three soluble sugars, fructose, glucose and sucrose, in both cultivars (Fig. 5). Initially, relatively low quantities (below 2 µmol mg−1 FW of the leaves) of all three sugars were recorded with no significant differences between treatments until 11 dai. Generally, cv. Stakado showed higher values for all sugars until 9 dai and a small peak occurred in sucrose accumulation at 7 dai, but there were no significant differences between treatments in either cultivar. From 11 dai, a drastic increase in the amount of all three sugars was recorded in cv. Sevin inoculated with S. tritici (to c. 110, 120 and 35 µmol mg−1 FW of the leaves for fructose, glucose and sucrose, respectively). Also a slight increase in sucrose concentration occurred in cv. Stakado inoculated with S. tritici at 13 dai (to c. 12 µmol mg−1 FW of the leaves).
Fig. 5 Mean content of soluble sugars in wheat (Triticum aestivum) cvs Sevin and Stakado either inoculated with Septoria tritici or treated with water. Values are means ± SE. (a) Fructose, (b) glucose, and (c) sucrose.
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Discussion We investigated the role of H2O2 in wheat infected by the hemibiotrophic pathogen S. tritici and show that infiltration with catalase in wheat leaves effectively scavenged early produced H2O2, thus resulting in decreased latent period (Fig. 1), increased penetration and mesophyll colonization (Tables 1, 2) and fungal biomass (Fig. 2) in both a susceptible and a resistant cultivar. By contrast, infiltration with H2O2 had the reverse effect, generally resulting in reduced fungal colonization and biomass and increased latent period. The initial changes in fungal biomass were, however, so small that significant differences could only be observed from 12 dai. Such clear changes in susceptibility by manipulation of a single defence compound such as H2O2 (both ways) using a simple and delicate approach has, to our knowledge, not been demonstrated before. The only known role for catalase is to scavenge H2O2, and, furthermore, the control (infiltration of heat-inactivated catalase) in cv. Sevin showed that it is the catalase enzyme activity and not perception of the catalase protein molecule per se that is responsible for the effect observed. Scavenging of H2O2 in the resistant cv. Stakado also resulted in enhanced pathogen growth in the substomatal cavities, but did not result in pycnidium formation, fungal growth most often stopping when reaching the mesophyll. Presumably, the reason for this is that this cultivar has several additional defence responses, in addition to H2O2, which contribute to pathogen arrest. Observed responses, which may contribute to resistance, include accumulation of peroxidase, β-1,3-glucanase, chitinase and callose (Shetty et al., 2003; N. P. Shetty, unpublished). However, another possible reason for the lack of a clear effect of catalase infiltration is that H2O2 concentrations in Stakado are so high, they cannot be depleted by catalase infiltration. A similar observation has been made in the barley–Blumeria graminis f. sp. hordei pathosystem, where it was not possible to scavenge all the H2O2 produced, despite external application of antioxidants (Zhou, 1998). In addition, fungal growth in our study was probably not affected by the infiltrated substances some days after the last infiltration because of their degradation, making it likely that the plants reverted to their ‘natural’ mode of interaction with the pathogen. Septoria tritici is generally classified as a hemibiotrophic pathogen with a long symptomless phase that is generally considered to be biotrophic. This is followed by a necrotrophic phase, which starts with tissue collapse and necrosis, later leading to pycnidium formation (Parbery, 1996; Shetty et al., 2003). Accumulation of H2O2 as a defence response that inhibits S. tritici during its initial biotrophic phase is in accordance with studies of other biotrophic pathogens that have been found to be inhibited by this ROS (ThordalChristensen et al., 1997; Mellersh et al., 2002). Since S. tritici shifts from a biotrophic to a necrotrophic lifestyle, H2O2
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could be anticipated to have a different role during the latter phase. Thus, necrotrophic pathogens have been reported to benefit from elevated H2O2 concentrations. For example, Govrin & Levine (2000) concluded that Botrytis cinerea infecting Arabidopsis thaliana induced accumulation of H2O2 in order to kill host cells to facilitate invasion. Likewise, it has been concluded that ROS were important for pathogenicity of other necrotrophic and hemibiotrophic pathogens such as Drechslera spp., Rhynchosporium secalis and Bipolaris sorokiniana (Gönner & Schlösser, 1993; Kumar et al., 2001; Able, 2003). A massive accumulation of H2O2 occurred in susceptible wheat infected by S. tritici during the late stages of the interaction (Shetty et al., 2003). If S. tritici induces the same pattern of H2O2 accumulation as necrotrophic pathogens, scavenging of H2O2 during this stage should make the plants more resistant and hinder pathogen colonization and symptom expression. However, surprisingly, scavenging of H2O2 with catalase increased pathogen growth and reduced the latent period, whereas infiltration of H2O2 had the reverse effect. This demonstrates that H2O2 is also harmful to S. tritici during the necrotrophic phase, thus contradicting previous reports on the role of H2O2 for necrotrophic pathogens. Among the several potential roles for H2O2 in defence (Lamb & Dixon, 1997; Neill et al., 2002), a direct antimicrobial effect could, among others, be envisaged in the wheat–S. tritici pathosystem. In vitro experiments demonstrated a direct inhibitory effect of 10 mM H2O2 on 4-d-old inoculum. Similarly, millimolar concentrations of H2O2 were found to inhibit pathogen growth in other host–pathogen systems (Peng & Kuc, 1992; Wu et al., 1995). The concentration of H2O2 applied by infiltration gave a strong and clear effect, though less than that which had an effect on fungal growth in vitro. The probable reason for this is that the H2O2 is highly localized in planta and the infiltration procedure is not able to mimic the timely and localized release of H2O2 occurring naturally in the plant in response to infection and spread of the pathogen. We found that the in vitro growth of the S. tritici harvested from 16-d-old cultures was inhibited less by H2O2 concentration than inoculum harvested from 4-d-old cultures. Obviously, it is difficult to relate changes observed in vitro with the in vivo changes, since the latter occur in a close interaction with the host. Nevertheless, it is interesting that this difference in sensitivity parallels the change in fungal physiology occurring during the transition from biotrophic to necrotrophic growth. An interesting observation from these experiments is that, whereas the initial stages of penetration by S. tritici were generally enhanced by the early infiltrations of catalase, H2O2 infiltrations often reduced them. These observations indicate that H2O2 has a very strong effect on penetration events. This is supported by the fact that H2O2 accumulation is particularly strong in the stomatal complexes during infection (Shetty et al., 2003), and that the pathogen is highly sensitive to H2O2 just after inoculation (cf. Fig. 3).
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An alternative explanation for the role of H2O2 in defence is that expression of host defence response genes is activated, which has also been observed (Ray et al., 2003, N. P. Shetty, unpublished), whereas classical HR (sensu Klement, 1982) has not been observed (Kema et al., 1996; Shetty et al., 2003). Collectively, our data demonstrate that H2O2 is very important in the defence of wheat against S. tritici during the biotrophic phase of the interaction and that the pathogen does not benefit from the H2O2 present during the necrotrophic phase, even though it can be tolerated (Shetty et al., 2003). This raises the question as to the origin of the massive, late accumulation of H2O2 and how the pathogen copes with it. We do not anticipate fungal origin of the H2O2, and the mitochondria are normally relatively unimportant ROS generators in photosynthesizing tissue (Foyer & Noctor, 2000; Apel & Hirt, 2004; Kuzniak & Sklodowska, 2005). A likely source is therefore the chloroplasts. Thus, if damage occurs to the chloroplasts, chlorophyll will be released and this chlorophyll will in turn release H2O2 and other ROS if it is not readily degraded by the plant (Kariola et al., 2005). Therefore, we examined whether photosynthesis was affected by inoculation with S. tritici. Photosynthesis in cv. Sevin inoculated with S. tritici was only down-regulated from 11 dai, corresponding with the first appearance of symptoms. This was, however, not related to a decrease in chlorophyll content in this cultivar even though the tissue became necrotic and H2O2 accumulated (Shetty et al., 2003). The reason for the increase in chlorophyll content per mg FW seen in cv. Sevin at 7 dai is not known, whereas the increase at 15 dai is the result of the reduced FW of the leaves caused by their wilting and necrosis. In conclusion, it appears less likely that the late H2O2 accumulation is the result of stress on photosynthesis. The other major source of ROS in photosynthesizing tissue is the peroxisomes, and it is likely that stress on these bodies is important. Thus, peroxisomes have been shown to be implicated in natural leaf senescence (Pastori & del Río, 1997; Distefano et al., 1999). Furthermore, Kuzniak & Sklodowska (2005) showed that B. cinerea infecting tomato induced stress on the peroxisomes, leading to collapse of their antioxidant system, thus partly explaining the pathogen-related leaf death. Whether the same explanation also applies for S. tritici remains to be elucidated. We are currently investigating the source of H2O2 in the wheat–S. tritici system. It has been shown that carbohydrates are involved as signalling compounds in down-regulation of photosynthesis (Wright et al., 1995; Ehness et al., 1997; Sinha et al., 2002), and increased sugar concentrations have been shown previously to increase host defence response gene expression following pathogen attack (Ehness et al., 1997). In the wheat– S. tritici interaction, we found that photosynthesis was downregulated from 11 dai and this was followed by release of H2O2 by 13 dai (Shetty et al., 2003). Therefore, we quantified fructose, glucose and sucrose to study if this could explain the effect on photosynthesis and ROS release. We found a very
large accumulation of all three sugars in cv. Sevin inoculated with S. tritici starting before initiation of the necrotrophic phase of the interaction (indicated by cell collapse, necrosis and increase in fungal biomass), with a small increase also occurring in cv. Stakado. The sugar accumulation pattern we observed is contrary to that reported for the necrotrophic pathogen B. cinerea (Berger et al., 2004), but is in accordance with observations made for the biotrophic pathogen Blumeria graminis f. sp. tritici (Wright et al., 1995). S. tritici will benefit from this late, massive sugar release as it feeds on apoplastic carbohydrates (Rohel et al., 2001). Nevertheless, Rohel et al. (2001) observed that during sporulation, there were indications of carbohydrate starvation in the fungus, suggesting a need to increase availability of nutrients. Therefore, when apoplastic carbohydrates are depleted, the pathogen probably stimulates sugar release to initiate pycnidium formation, thereby eliciting host tissue necrosis. Sugar release could also be the consequence of toxin production by the pathogen (Shetty et al., 2003). Sugar release could result in host tissue necrosis and the reduction of photosynthesis, or, alternatively, stress on the peroxisomes could trigger subsequent ROS release. Tissue necrosis will result in activation of defence reactions, releasing H2O2, which could participate in cell death and reduction of photosynthesis. In support of this suggested chain of events is the observation that, in cv. Sevin, S. tritici lives practically unnoticed until sporulation, at which time defence responses are finally expressed (Shetty et al., 2003; N.P. Shetty, unpublished). Alternatively, as a consequence of the repression of photosynthesis, the host may react, at the onset of symptom expression, by changing to heterotrophic metabolism (Ehness et al., 1997), or assimilates from the chloroplasts may be translocated to other parts of the plant as a response to stress (Krupinska & Humbeck, 2004). Irrespective of the source, the late H2O2 accumulation probably stimulates the pathogen to enter its necrotrophic phase as a requirement to survive. The pathogen thus copes with the toxic substances accumulating at this stage and survives the death of host tissues. During the initial, biotrophic stage, the pathogen does not penetrate any cell (Cohen & Eyal, 1993; Kema et al., 1996; Shetty et al., 2003) and it survives, presumably by attempting to avoid recognition until it has produced a sufficient amount of biomass to initiate reproduction. In this respect, we can speculate whether the symptomless phase is really biotrophic sensu stricto, or whether the pathogen is, in fact, an endophyte, that is, living saprohytically and not pathogenically. In further support of this proposition is the fact that S. tritici lives strictly in the apoplast with no special intracellular feeding structures (Palmer & Skinner, 2002).
Acknowledgements We thank Dr Lisbeth Mikkelsen for critically reviewing the manuscript. Sejet Plant Breeding and Abed Fonden are
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acknowledged for providing seeds of cvs Sevin and Stakado, respectively. This work was supported by the Danish Research Council for Technology and Production Sciences (grant ‘Understanding the role of ROS in defence and pathogenesis in the wheat–S. tritici interaction. How does the pathogen manipulate the host for successful infection?’)
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