Production of hydrogen peroxide and expression of ROS-generating genes in peach flower petals in response to host and non-host fungal pathogens




Reactive oxygen species (ROS) play dual roles in plant–microbe interactions in that they can either stimulate host resistance or enhance pathogen virulence. Innate resistance in peach (Prunus persica) to the brown rot fungal pathogen Monilinia fructicola is very limited, and knowledge of the mechanism of virulence is rudimentary. In this study, production of hydrogen peroxide, a major component of ROS, was determined in peach flower petals in response to M. fructicola (a host pathogen) and Penicillium digitatum (a non-host pathogen). Monilinia fructicola was able to infect flower petals while P. digitatum was not. During the host-specific interaction, M. fructicola induced hydrogen peroxide accumulation in flower petals. Application of exogenous antioxidants significantly reduced hydrogen peroxide accumulation as well as the incidence of brown rot disease. Application of M. fructicola spores to the surface of intact flower petals induced gene expression and increased enzyme activity of NADPH oxidase and cell wall peroxidase in host tissues, resulting in the production of hydrogen peroxide. Petals inoculated with M. fructicola exhibited high levels of protein carbonylation and lipid peroxidation. No significant response in gene expression, enzyme activity or hydrogen peroxide levels was observed in peach flower petals treated with P. digitatum. These results suggest that M. fructicola, as with other necrotrophic fungi, uses the strong oxidative response as part of a virulence mechanism.


Monilinia fructicola is one of the most devastating pathogens of Prunus species, including peach, nectarine, plum and cherry (Lee & Bostock, 2007). It causes blossom and twig blight, and brown rot decay in fruit. Susceptibility of peach to M. fructicola is dependent on the stage of annual development. Bloom period is one of the most susceptible stages, with petals serving as a prime infection court. Latent infections on developing fruit can also result in severe yield losses (Lee & Bostock, 2006). Currently, brown rot disease management in stone fruits is mainly based on the use of fungicides; however, resistance is a growing problem (Luo & Schnabel, 2008). A better understanding of the biology of M. fructicola pathogenesis could provide opportunities for new control strategies and the development of resistant cultivars.

An oxidative burst in host tissues, consisting of a two-phase production of hydrogen peroxide, is believed to present the first line of defence in numerous plant–pathogen interactions and has been documented to be an aspect of many resistant interactions (Nanda et al., 2010). In contrast, failure of the plant host to develop a rapid and massive accumulation of reactive oxygen species (ROS) in response to an invading pathogen usually results in infection, a phenomenon observed in many compatible host–bacterial interactions (Pauly et al., 2006). The role of host-generated ROS in fungal virulence is more obscure, as different or even conflicting findings have been reported. Some studies have suggested that necrotrophic pathogens, such as Botrytis cinerea and Sclerotinia sclerotiorum, benefit from the host oxidative burst because fungal growth and symptom development are positively correlated with levels of hydrogen peroxide (H2O2), a major component of ROS (Govrin & Levine, 2000). However, for the same pathogens, the ability to suppress a defence-related oxidative burst in host tissue has been shown to be critical for developing an infection (Cessna et al., 2000). Biotrophic pathogens are generally considered to be more sensitive to ROS than necrotrophic species, especially at the initial stages of interaction with a host plant. However, at consecutive steps of host invasion, biotrophs also become less vulnerable to, or even benefit from, host-produced ROS (Glazebrook, 2005). The basis for this selective receptivity to host oxidative radicals by pathogenic fungi is unknown.

A previous study on the role of ROS on M. fructicola pathogenicity indicated that host phenols can influence intracellular antioxidant levels in the pathogen, and that changes in the redox environment may influence both gene expression and the development of structures used by the pathogen to facilitate infection (Lee & Bostock, 2007). A more recent study reported that exposure to H2O2 increased expression of cutinase transcripts in M. fructicola in vitro, suggesting that increased ROS levels in host tissue would enhance pathogen virulence as well (Lee et al., 2010). However, to date, no research has examined the production of ROS in Prunus spp. in response to M. fructicola infection. The objective of the present study was to evaluate the production of H2O2 in peach petals in response to inoculation with spores of M. fructicola (a host pathogen) and Penicillium digitatum (a non-host pathogen). Both species are considered necrotrophic pathogens. An analysis of the effect of exogenous antioxidants and changes in host redox enzymes in both host-specific and non-host-specific interactions was also conducted.

Materials and methods

Fungal pathogens

Monilinia fructicola was isolated from brown rot infected peach fruit, and P. digitatum was from a laboratory stock culture originally obtained from infected citrus fruit. Both spore suspensions were obtained from 10-day-old cultures on potato dextrose agar (PDA; Difco) at 25°C. The number of spores was calculated using a Cellometer Vision (Nexcelom Bioscience), and the spore concentration was adjusted to 1 × 105 spores mL−1 with phosphate-buffered saline (PBS, pH 6·0).

Flower petals and inoculation

Fully expanded flowers were collected from mature peach trees (Prunus persica cv. Harrow Beauty) in an orchard on the grounds of the USDA-ARS-Appalachian Fruit Research Station, Kearneysville, WV, USA. Flowers without injury or disease were selected based on uniformity of size, disinfected with 0·05% sodium hypochlorite for 1 min, and rinsed in sterile water. Petals were gently removed from the flowers and washed with sterile water once more. Under sterile conditions, clean petals were placed in Petri dishes lined with wet filter paper. Inoculation of the flower petals was performed as described previously (Lee & Bostock, 2007). Each petal was inoculated with either M. fructicola or P. digitatum by placing 5 μL of a spore suspension (1 × 105 spores mL−1) in PBS on the centre of the petal. Petals inoculated with PBS alone served as a control. Observations of pathogen development on peach petals were conducted at 8 and 20 h post-inoculation (hpi) at 25°C using a Zeiss Axiophot (Carl Zeiss) microscope. Disease symptoms, if any, were recorded at 20 hpi. For each experiment, a treatment consisted of three replicates with nine petals per replicate and the experiment was repeated three times.

Hydrogen peroxide detection in peach petals in response to pathogens

The oxidant-sensitive fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen) was used to assess H2O2 production in plant tissue (Ortega-Villasante et al., 2007). At 8 and 20 hpi, the spore–PBS suspension (1 × 105 spores mL−1) of P. digitatum or M. fructicola in either 1 mm l-ascorbic acid or citric acid was removed with a pipette from the surface of treated flower petals. Then, 20 μL of PBS buffer (pH 6·0) containing 10 μm H2DCFDA was applied over and beyond the location where the inoculation drops were administered. After 30 min incubation in darkness at 30°C, petals were washed twice with PBS buffer and examined on a Typhoon Trio (GE/Amersham Biosciences) scanner using a blue laser (488 nm excitation and 520 nm emission at 300 V). After imaging the petals on the Typhoon Trio, fluorescent-positive petals were examined with the Axiophot microscope with a UV-light source. For each experiment, a treatment consisted of three replicates with nine petals per replicate and the experiment was repeated three times.

Effects of antioxidants on growth of M. fructicola in vitro

The effects of l-ascorbic acid or citric acid (Sigma-Aldrich) on spore germination and mycelial growth of M. fructicola in vitro was determined as described previously (Liu et al., 2010). Aliquots of 100 μL spore suspension at 1 × 105 spores mL−1 were plated on Petri dishes (90 mm diameter) with 20 mL PDA (made with PBS, pH 6·0) containing 0, 0·1, 0·5, 1, 5 or 10 mm l-ascorbic acid or citric acid. Petri dishes were incubated at 25°C for 5 h. Germination was assessed on approximately 200 spores per treatment within each replicate.

The mycelial disks (5 mm diameter) from 10-day-old cultures of M. fructicola were placed in the centre of Petri dishes (90 mm diameter) containing 20 mL PDA with different concentrations of l-ascorbic acid or citric acid (0, 0·1, 0·5, 1, 5 and 10 mm), then incubated at 25°C. Mycelial growth was characterized by measuring colony diameter 5 days after inoculation. Each treatment was replicated three times and the experiment was repeated three times.

Effects of antioxidants on brown rot caused by M. fructicola in petals

In order to investigate whether antioxidants could alleviate the disease symptom of brown rot, flower petals were inoculated with 5 μL of M. fructicola spores (1 × 105 spores mL−1) in PBS (pH 6·0) containing 1 mm l-ascorbic acid or citric acid. The concentration of acid was based on preliminary data indicating that this concentration did not have a direct inhibitory effect on the in vitro growth of M. fructicola. Inoculation of petals with a suspension of M. fructicola spores in PBS without antioxidants served as a control. Disease symptoms in petals were recorded 20 hpi. Additionally, to evaluate the effect of the phenolic antioxidants caffeic and chlorogenic acid on brown rot lesion development in peach flower petals, 5 μL spore suspension (1 × 105 spores mL−1 in PBS) containing either 1 mm caffeic or chlorogenic acid was pipetted onto the surface of a flower petal, while 5 μL of a spore suspension without the test compounds was placed on the opposite side of the petal. Each experiment consisted of nine petals per treatment replicated three times and the entire experiment was repeated three times.

RNA isolation and semiquantitative RT-PCR analysis of NADPH oxidase (nox) and cell wall peroxidase (pod) expression

Total RNA was isolated from peach petal samples at several time points (0, 8 and 20 hpi) using an RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer's instructions. Extracted RNA was treated with TURBO DNase (Ambion) and purified again with RNeasy. Aliquots of 1 μg total RNA were used for first strand cDNA synthesis in 20 μL reaction volume with 100 units of M-MLV reverse transcriptase (Ambion). Transcript levels of tubulin-α (NCBI accession no. DY650410; forward primer 5′-AGATGCCCAGTGATGCCTCAG-3′; reverse primer 5′-ACCAGTACCACCACCAACAGC-3′) in peach served as an internal control gene (Li et al., 2009). Cycling parameters for each gene amplification were 95°C for 5 min; 25 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s; and finally 72°C for 10 min. Information taken from the Genome Database for Rosaceae (GDR; about peach gene sequences of nox (GDR Landmark: ppa000913m; forward primer 5′-TCCTCCGCGCCGATTTTGCT-3′; reverse 5′-CAAAGTCTCGGCAGCGCCCT-3′) and pod (GDR Landmark: ppa023604m; forward primer 5′-AGCAGAGCAGTTTCGCAAGAACG-3′; reverse 5′-CAGCGGCGTCTGCGAGTAGTTG-3′) was used for primer design for PCR amplification. PCR products were cloned, sequenced to verify their identity, and deposited in NCBI GenBank (accession nos: nox, JN791439; pod, JN791440). Quantification of transcript expression level was based on the band intensity on 2% (w/v) agarose gels. There were three replicates in each treatment and the experiment was repeated three times.

Assay for enzyme activity

Plasma membrane from petal cells was isolated as described previously (Xia et al., 2009). The NADPH oxidase (NOX) activity in isolated plasma membranes was examined using superoxide dismutase (SOD)-inhibitable ferricytochrome c reduction. An aliquot of the isolated plasma membrane was added to a reaction mixture consisting of 50 mm HEPES-KOH (pH 7·8), 100 mm EDTA, 50 mm ferricytochrome c and 100 mm NADPH in the presence or absence of SOD (200 units mL−1, SOD from bovine erythrocytes; Sigma-Aldrich) and incubated at room temperature for 30 s. NOX activity was measured by monitoring the cytochrome c reduction at 550 nm, and expressed as U mg−1 protein, where one unit of enzyme was defined as the amount necessary to generate 1 μm superoxide anion per min.

Cell wall peroxidase (POD) activity was determined by the method described previously (Xue et al., 2008) with a slight modification. Flower petals (1 g) were homogenized in 3 mL of 10 mm potassium phosphate buffer (pH 6·4). After centrifugation (2000 g, 4°C, 6 min), the supernatant was discarded and the pellet was washed with the same volume of extraction buffer and centrifuged in the same way five times. The resulting pellet was then incubated in 1 m NaCl with shaking for 2 h and centrifuged at 13 000 g for 15 min at 4°C. The supernatants were used as cell wall-bound enzyme samples. POD activity was assayed by measuring the oxidation rate of the substrate coniferyl alcohol at 260 nm in a reaction mixture consisting of 100 mm sodium phosphate buffer (pH 7·0), 0·1 mm coniferyl alcohol, 0·5 mm H2O2 and enzyme extract. POD activity was expressed as U mg−1 protein, where one unit of enzyme was defined as the amount necessary to decompose 1 μm of substrate per min.

Protein content was measured using the Bradford assay, with bovine serum albumin (Sigma-Aldrich) as a standard (Bradford, 1976). There were three replicates in each treatment, and the experiment was repeated three times.

Determination of protein carbonylation and lipid peroxidation

Petal samples were disrupted in liquid nitrogen by grinding with a plastic pestle. Protein oxidation was measured by determining carbonyl content (Hoque et al., 2008). The carbonyl content was expressed as nmol mg−1 protein. For assaying lipid peroxidation, the method based on the reaction of thiobarbituric acid with reactive species derived from lipid peroxidation, particularly malondialdehyde (MDA) was used. Detection of thiobarbituric acid-reactive species was carried out by a colorimetric assay (Ritter et al., 2008). The lipid peroxidation was expressed as nmol MDA mg−1 protein.

Protein content was measured as described previously (Bradford, 1976). There were three replicates in each treatment, and the experiment was repeated three times.

Data analysis

All statistical analyses were performed with spss v. 13.0 (SPSS Inc.). Data were analysed by one-way anova. Mean separations were performed by Duncan's multiple range tests. Differences at < 0·05 were considered to be significant. Data presented in this paper were pooled across three independent repeated experiments, as the interaction between treatment and experimental replication was not significant.


Development of M. fructicola and P. digitatum on peach petals

The development of M. fructicola and P. digitatum on peach petals is shown in Figure 1. Appressoria of M. fructicola were observed on petals at 8 hpi (Fig. 1a). Hyphae of M. fructicola were observed growing within petal tissues at 20 hpi (Fig. 1b). In contrast, spore germination of P. digitatum, a non-host pathogen, on peach petals was not detected at 8 hpi (Fig. 1c). By 20 hpi only sporadic germination of P. digitatum was observed on the petal surface (Fig. 1d). No indication of P. digitatum hyphae within petal tissues was ever observed.

Figure 1.

Monilinia fructicola (a,b) and Penicillium digitatum (c,d) germination and development on peach flower petals at 8 (a,c) and 20 h (b,d) post-inoculation (hpi). The inoculated peach petals were stained with lactophenol cotton blue prior to observation. Scale bar represents 20 μm. S, spore; A, appressoria; H, hyphae.

Disease symptoms and hydrogen peroxide accumulation in peach petals in response to M. fructicola and P. digitatum

Peach petals inoculated with PBS or P. digitatum did not exhibit any disease symptoms, whereas inoculation with M. fructicola caused necrosis at sites of inoculation with 100% incidence at 20 hpi (Fig. 2). Accumulation of H2O2 in and around necrotic areas induced by the brown rot pathogen was detected with the fluorescent probe H2DCFDA (Fig. 3). In contrast, PBS and P. digitatum did not induce any H2O2 accumulation in petals. H2O2 accumulation increased in M. fructicola-inoculated petals over the observed time span of 8–20 hpi. The accumulation of H2O2 appeared to be initially localized in the cell walls of petal cells but later appeared to be generally distributed (see magnified inserts in Fig. 3). The addition of the antioxidant compounds, l-ascorbic acid or citric acid, to the inoculation droplet decreased H2O2 accumulation. At 8 hpi, no H2O2 accumulation was observed in petals inoculated with M. fructicola + 1 mm l-ascorbic acid or citric acid. At 20 hpi, the level of H2O2 detected in petals inoculated with M. fructicola + 1 mm l-ascorbic acid or citric acid was significantly lower than in petals that had been inoculated with M. fructicola alone.

Figure 2.

Disease symptom in peach petals inoculated with PBS, Penicillium digitatum (Pd) or Monilinia fructicola (Mf) at 20 h post-inoculation. Scale bar represents 1 cm. DI, disease incidence.

Figure 3.

H2O2 accumulation in peach petals inoculated with PBS, Penicillium digitatum (Pd), Monilinia fructicola (Mf), Mf + 1 mm l-ascorbic acid (AC), or Mf + 1 mm citric acid (CC) at 8 and 20 h post-inoculation (hpi). Photographs at the far right are magnifications of the H2O2-positive areas indicated by the arrows. The magnified areas illustrate that H2O2 was initially localized to the cell walls of petal cells but later became more generally distributed. RFI, relative fluorescence intensity.

Effect of antioxidants on growth of M. fructicola in vitro and in planta

Because l-ascorbic acid and citric acid inhibited host H2O2 accumulation when inoculated with M. fructicola, the effect of the antioxidants on M. fructicola was investigated. The effect of 0·1–10 mm l-ascorbic and citric acid on spore germination and mycelial growth of M. fructicola in vitro is shown in Figure 4. A small but significant inhibition of spore germination was observed at the highest concentration (10 mm) of l-ascorbic acid but no inhibition was observed at any of the tested concentrations of citric acid (Fig. 4a). Similar to the effect on germination, 10 mm l-ascorbic acid also inhibited mycelial growth of M. fructicola slightly (Fig. 4b). Mycelial growth was slightly inhibited by 5 and 10 mm citric acid. Based on these results and a prior study (Bostock et al., 1999) showing no inhibitory effect of ≤5 mm chlorogenic and caffeic acids on M. fructicola spore germination and growth, a concentration of 1 mm of these compounds was used to evaluate their effect as antioxidants on disease symptoms and pathogen development on inoculated peach petals.

Figure 4.

Effect of antioxidants, l-ascorbic acid and citric acid, on spore germination and mycelial growth of Monilinia fructicola (Mf). Error bars indicate standard deviations of the means (n = 9). Columns with different letters indicate significant differences according to Duncan's multiple range test (P < 0·05).

As measured at 20 hpi, both phenolic (chlorogenic and caffeic; Fig. S1) and non-phenolic (l-ascorbic and citric) acids at 1 mm significantly decreased disease incidence in petals caused by M. fructicola (Fig. 5). Disease incidence of petals inoculated with M. fructicola suspended in l-ascorbic or citric acid were 40% and 45%, respectively, while the incidence in petals inoculated with M. fructicola in PBS reached 100% (Fig. 5).

Figure 5.

Disease symptoms in peach petals inoculated with (a) 5 μL Monilinia fructicola at 1 × 105 spores mL−1; (b) M. fructicola + 1 mm l-ascorbic acid; and (c) M. fructicola + 1 mm citric acid. The disease incidence in each treatment was recorded at 20 h post-inoculation (d). The pH values in all treatments were 6·0 in PBS. Data presented are the means of pooled data. Error bars indicate standard deviations of the means (n = 9). Columns with different letters indicate significant differences according to Duncan's multiple range test (P < 0·05). Mf, M. fructicola; AC, l-ascorbic acid; CC, citric acid.

Gene expression and enzyme activity of NADPH oxidase (nox) and cell wall peroxidase (pod) of peach petals in response to M. fructicola and P. digitatum

The transcript level of two ROS-generating genes, nox and pod, increased during the experimental period (20 hpi) in petals inoculated with M. fructicola (Fig. 6). Elevated levels of nox and pod gene expression in M. fructicola-inoculated petals were evident at both 8 and 20 hpi. In contrast, nox and pod expression in PBS- and P. digitatum-treated peach petals did not increase but rather remained stable over the observed time period.

Figure 6.

The gene expression of NADPH oxidase and cell wall peroxidase of peach petal in response to PBS, Penicillium digitatum (Pd) and Monilinia fructicola (Mf). The tubulin-α gene was used as a control for normalizing mRNA quantity. The level of target gene expression relative to the sample at time 0 is shown above each band. Data presented are the means of pooled data (n = 9).

The activity of both NOX and POD enzymes in treated peach petals corresponded to the pattern of gene expression for these ROS-generating genes (Fig. 7). The highest level of NOX and POD enzyme activity was observed in M. fructicola-inoculated petals at 20 hpi while remaining comparatively low in PBS- and P. digitatum-treated peach petals.

Figure 7.

The enzyme activity of (a) NADPH oxidase (NOX) and (b) cell wall peroxidase (POD) of peach petal in response to PBS, Penicillium digitatum and Monilinia fructicola. Data presented are the means of pooled data. Error bars indicate standard deviations of the mean (n = 9).

Oxidative damage of protein and lipid in peach petals

Because elevated levels of H2O2 had been observed in M. fructicola-treated peach petals, the effect of this on oxidative damage to proteins and lipids was monitored. Carbonyl content (a measure of protein oxidation) and MDA content (a measure of lipid peroxidation) were both determined (Fig. 8). The levels of carbonyl and MDA content increased over the experimental period which corresponded with the increase in the level of ROS (Fig. 3). At time 0, prior to pathogen inoculation, carbonyl (Fig. 8a) and MDA (Fig. 8b) levels were low. As the time of incubation increased from 0 to 8 and 20 hpi, carbonyl and MDA levels increased in all the three treatments; however, the greatest increase by far was in the M. fructicola-treated samples.

Figure 8.

Protein carbonylation (a) and lipid peroxidation (b) in peach petal in response to PBS, Penicillium digitatum and Monilinia fructicola. Data presented are the means of pooled data. Error bars indicate standard deviations of the mean (n = 9).


This study was conducted to gain further insight into the redox environment in peach flower petals in response to M. fructicola infection. Susceptibility to brown rot is common to most, if not all, cultivars of peach. However, Feliciano et al. (1987) reported a significant level of brown rot resistance in the cultivar Bolihna. Compared to susceptible cultivars, Bolihna exhibited greater concentrations of phenolic acids in the fruit epidermis and subtending cell layers. Bostock et al. (1999), in a study of brown rot infection of peach fruit, reported that the concentration of the phenolic compounds, chlorogenic and caffeic acid, while initially high, declined during fruit development which coincided with an increase in susceptibility to M. fructicola. They suggested that chlorogenic and caffeic acids inhibit the production of M. fructicola cutinases rather than being directly toxic to the pathogen. While the chelation of iron by these phenolic acids was initially proposed as the mechanism of cutinase suppression in the pathogen, the antioxidant activity of caffeic acid was later reported to be more important (Wang et al., 2002). It has also been reported that the antioxidants chlorogenic acid, caffeic acid and reduced glutathione suppressed the production of cutinase and polygalacturonase in M. fructicola, and inhibited appressoria formation and subsequent lesion development in flower petals (Lee & Bostock, 2007). Collectively, these studies suggest that antioxidants suppress the expression of virulence factors in M. fructicola and interfere with the infection process. To determine if there are actual pathogen-induced changes in the redox environment in planta that can influence subsequent disease development, the current study evaluated H2O2 production in peach petals inoculated with M. fructicola (a host pathogen) versus P. digitatum (a non-host pathogen).

Within 20 hpi, M. fructicola induced necrotic lesions in all peach petals. Microscopy revealed that germinated spores developed appressoria as early as 8 hpi after inoculation. By 20 hpi, the brown rot pathogen developed vigorous hyphae growing under the cuticle of epidermal cells and within the petal tissue. These observations are in agreement with the previous report that M. fructicola predominantly infected Prunus spp. petals through the penetration of the cuticle from appressoria (Lee & Bostock, 2006). The subcuticular growth of M. fructicola versus the surface growth of P. digitatum may explain why P. digitatum (a necrotrophic pathogen like M. fructicola) did not induce necrotic lesions similar to those observed in M. fructicola-inoculated petals even though the spores of P. digitatum did germinate. The germination of P. digitatum spores placed on peach petal surface was surprising in itself because P. digitatum is a host-specific pathogen, and spores of this fungus typically germinate only in the presence of host-specific volatiles and nutrients (Droby et al., 2008). As intact peach petals have low levels of available nutrients compared to fruit tissues, a potential signal for P. digitatum spore germination may have been due to volatile compounds. There is an overlap between the volatiles emitted from Prunus and Citrus spp. (Kesselmeier & Staudt, 1999), therefore some of the volatile compounds (e.g. linalool) may potentially trigger the germination of P. digitatum spores in the absence of nutrients. However, additional studies will be needed to determine the validity of this hypothesis.

Inoculation of peach petals with M. fructicola resulted in a gradual accumulation of H2O2 over the experimental time period at sites of the inoculation droplet (Fig. 3). Inoculation with the same concentration of P. digitatum spores did not induce H2O2 production within inoculated areas of peach petals. This is the first experimental data to show that the brown rot pathogen M. fructicola induces accumulation of H2O2 in host tissues during invasion of the tissue. This finding correlates well with a previous study where paraquat treatment (known to generate high levels of H2O2) of stone fruit triggers the rapid stimulation and development of brown rot decay from previously established quiescent infections of M. fructicola (Northover & Cerkauskas, 1994).

To determine if the suppression of host oxidative response correlates with reduced virulence, the effect of non-phenolic antioxidants (ascorbic and citric acid) on H2O2 accumulation and symptom development in peach inoculated with M. fructicola was evaluated. Exogenous ascorbic and citric acids are suppressors/chelators of plant-produced H2O2 (Macarisin et al., 2007). The addition of either 1 mm ascorbic or citric acid inhibited H2O2 accumulation (Fig. 3), as well as symptom development caused by M. fructicola, without having direct antifungal activity (Figs 4 and 5). A previous study (Lee & Bostock, 2007), as well as the present results (Fig. 1), demonstrated that caffeic and chlorogenic acid also reduced brown rot lesion development in peach petals. Considering that both phenolic and non-phenolic antioxidants had a similar effect in inhibiting disease symptoms, inhibition of the infection process may have been primarily due to the suppression of the oxidative response in the host.

Importantly, H2O2 production was first observed in the cell walls of the flower petal (Fig. 3), supporting the premise that enzymes bound to the cell wall or plasma membrane may be responsible for the initial production of ROS (Torres et al., 2006) during the M. fructicola–peach interaction. Furthermore, increased levels of gene expression and enzyme activity were detected for both NADPH oxidase (NOX) and cell wall peroxidase (POD). NOX, known as the respiratory burst oxidase, catalyses the production of inline image by the one-electron reduction of molecular oxygen using NADPH as an electron donor (Brown & Griendling, 2009). The rapid transformation of inline image into H2O2 during plant–pathogen interactions was first observed in potato tubers infected with Phytophthora infestans (Doke, 1983). Rboh (respiratory burst oxidase homologues) genes encoding gp91phox (the catalytic subunit of the NOX of phagocytes) have been reported in the majority of plants (Zurbriggen et al., 2010). Pathogen-induced, NOX-derived ROS was demonstrated to play a role in suppressing the spread of cell death in Arabidopsis (Torres & Dangl, 2005). In addition to NOX, POD has been proposed as another producer of H2O2 generation during the initial stage of a host–pathogen interaction (Bindschedler et al., 2006). For example, POD was proposed to be the major source of ROS in suspension-cultured cells of French bean (Phaseolus vulgaris) treated with a cell wall elicitor from Colletotrichum lindemuthianum (Bolwell et al., 1998). The results of the present study showed that M. fructicola increased nox and pod gene expression in peach petals (Fig. 6), as well as the activity of their respective enzymes (Fig. 7). Similar inductive effects by pathogens have been reported on other host plants. Nox and nitric oxide associated 1 (noa1) has been shown to play a significant role in the oxidative burst in Nicotiana benthamiana leaves inoculated with P. infestans (Asai et al., 2008), while pod in pepper (Capsicum annuum) was reported to be involved in ROS generation and the activation of defence responses in response to invasion by bacterial pathogens (Choi et al., 2007). However, the present study is the first report demonstrating the inductive effect of M. fructicola on both gene expression and enzyme activity of both NOX and POD.

Increased levels of protein and lipid oxidation occur in cells exhibiting ROS accumulation, and have a deleterious effect on cellular structure and function. The level of carbonyl groups of proteins is widely used as a marker for oxidative damage to proteins (Stadtman & Levine, 2003) while MDA content serves as a marker for lipid peroxidation (Du & Bramlage, 1992). Higher carbonyl and MDA levels were detected in M. fructicola-treated petals compared to PBS and P. digitatum treatments at both 8 and 20 hpi (Fig. 8). The higher level of carbonylation and MDA may be directly related to the H2O2 accumulation observed in the plant host during pathogen infection. The data indicate that M. fructicola-induced stimulation of host H2O2 and the resulting oxidative damage in host tissues may partially explain the observed necrosis in peach petals.

In conclusion, the application of M. fructicola spores to the surface of peach flower petals triggered an accumulation of H2O2 in host tissues, potentially by activating host NOX and POD. H2O2 accumulation subsequently caused oxidative damage to proteins and lipids in the peach host. This chain of events may play a major role in the successful infection and resulting necrosis of peach tissues by M. fructicola. This premise is supported by the observation that suppression of ROS by the application of antioxidant compounds in a concentration that did not affect spore germination inhibited or prevented infection. The understanding of the role of ROS in the pathogenesis of M. fructicola has implications for developing management strategies for this major disease of stone fruit.


This research was supported by a grant (IS-4268-09) from the United States–Israel Binational Agricultural Research and Development (BARD) Fund to SD and MW.