Exogenous melatonin improves Malus resistance to Marssonina apple ...

J. Pineal Res. 2013; 54:426–434

© 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/jpi.12038

Journal of Pineal Research

Exogenous melatonin improves Malus resistance to Marssonina apple blotch Abstract: We examined whether exogenously applied melatonin could improve resistance to Marssonina apple blotch (Diplocarpon mali) by apple [Malus prunifolia (Willd.) Borkh. cv. Donghongguo]. This serious disease leads to premature defoliation in the main regions of apple production. When plants were pretreated with melatonin, resistance was increased in the leaves. We investigated the potential roles for melatonin in modulating levels of hydrogen peroxide (H2O2), as well the activities of antioxidant enzymes and pathogenesis-related proteins during these plant–pathogen interactions. Pretreatment enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhance the activities of plant defence-related enzymes, possibly improving disease resistance. Because melatonin is safe and beneficial to animals and humans, exogenous pretreatment might represent a promising cultivation strategy to protect plants against this pathogen infection.

Lihua Yin, Ping Wang, Mingjun Li, Xiwang Ke, Cuiying Li, Dong Liang, Shan Wu, Xinli Ma, Chao Li, Yangjun Zou and Fengwang Ma State Key Laboratory of Crop Stress Biology in Arid Areas/College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China Key words: disease resistant, Malus, Marssonina apple blotch, melatonin, pathogenesis-related proteins, ROS Address reprint requests to Fengwang Ma, Department of Pomology, College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China. E-mails: [email protected] & fwm64@nwsuaf. edu.cn Received November 19, 2012; Accepted December 21, 2012.

Introduction Marssonina apple blotch, caused by the fungus Diplocarpon mali [Y. Harada & K. Sawamura (anamorph Marssonina coronaria (Ell. & J. J. Davis) J. J. Davis, syn. Marssonina mali (Henn.) S. Ito)] [1], is one of the most severe apple diseases [2]. This disease leads to defoliation during the growing season, thereby weakening tree vigor and diminishing fruit yield and quality [3]. The fungus mainly infects leaves but can also attack twigs and fruits [4]. This disease is first manifested by brown spots and dark green circular patches on the leaf upper surfaces in mid-Summer. As it progresses, those spots coalesce and black pinhead-like asexual fruiting bodies develop on the affected tissue. Severe infections result in leaves turning yellow, followed by premature defoliation. This disease has been recorded in Canada, Japan, Brazil, China, Korea, India, and Italy [1, 5–9]. Usually occurring in consecutive years, it has become a serious problem within major regions of apple production [10]. Plants have developed sophisticated mechanisms to protect themselves against pathogen infections. Their immunity can be triggered by pattern-recognition receptors (PRRs) that act via pathogen-associated molecular patterns (PAMP) and effectors. These outcomes are designated as PAMP-triggered immunity (PTI) or effector-triggered immunity (ETI) [11]. This recognition leads to defence responses, such as oxidative bursts, transcriptional induction of pathogenesis-related genes, and the deposition of callose to strengthen the cell wall at sites of infection [12]. Oxidative burst, which involves the production of reactive oxygen species (ROS), is a nearly ubiquitous response of plants to pathogen attack following successful pathogen recognition. ROS has been proposed as orchestrating the 426

establishment of these defence responses [13]. ROS-scavenging systems have an important role in regulating the amount of ROS that is generated [14]. Chitinase and b-1,3glucanase, two pathogenesis-related proteins that are the downstream components of defence signaling, have direct antimicrobial activities [15]. Moreover, secondary metabolites, such as phenolic compounds, are directly involved in the plant response to pathogens. They are either toxic to pathogens or can be deposited inside the cell wall as an important first line of defense against infection [16]. Melatonin (N-acetyl-5-methoxytryptamine) is ubiquitous in living organisms. It exhibits pleiotropic biological activities in species from bacteria to mammals [17]. In mammals, melatonin is a natural antioxidant with immunity-enhancing properties [18]. In plants, it acts as a growth regulator, similar to the role of indole acetic acid, in directing the differentiation of cells, tissues, and organs [19]. Studies have mainly focused on the ability of melatonin to alleviate the effects of abiotic stresses such as heavy metals [20], UV radiation [21], temperature fluctuations [22], drought resistance [23], and high salinity conditions [24]. This compound can also delay leaf senescence [25, 26]. However, little is known about its function in response to biotic stresses. As a precursor, serotonin has a role in rice defences against Bipolaris oryzae [27]. Vitalini et al. [28] have shown that treating grape plants with resistance inducers can increase the contents of both melatonin and total polyphenols in the resulting wine compared with levels measured from products of the untreated control. However, many questions remain unanswered: (i) Whether exogenous applications of melatonin can protect plants from pathogen infection; (ii) whether melatonin modulates ROS levels in the plant–pathogen interaction, based on knowledge from a previous study [29] that it acts as a free radical scavenger and broad-spectrum antioxidant; and (iii)

Melatonin improves disease resistance whether melatonin can promote activities of pathogenesisrelated proteins during those plant–pathogen interactions. Here, we used the pathosystem of Malus–D. mali to determine whether pretreatment with melatonin could alleviate the damage caused by this pathogen. We also explored how melatonin, in its role as protector against Marssonina apple blotch, might possibly affect apple plants. We monitored changes in activities of several antioxidant enzymes, that is, catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), and guaiacol peroxidase (POD; EC 1.11.1.7) as well as of enzymes involved in the plant defences, including phenylalanine ammonia-lyase (PAL; EC 4.1.3.5), chitinase (EC 3.2.1.14), and b-1,3-glucanase (EC 3.2.1.39). Our goal was to determine any possible involvement by melatonin-induced protection against this disease. Our theory was that, if melatonin functions as a defence compound, it may be used within a feasible agricultural approach to protect plants from pathogen infections.

Materials and methods Plant material We used plants of Malus prunifolia (Willd.) Borkh. cv. Donghongguo identified in our previous study (unpublished data) as being susceptible to Diplocarpon mali. This valuable species is hosted in China, where it is mainly used as an apple rootstock but is also cultivated for fruit production. For the current investigation, cultivar Donghongguo was grafted onto M. hupehensis (Pamp.) Rehd. Two-year-old grafted trees were grown in a greenhouse at the College of Horticulture, Northwest A and F University, Yangling (34°20′N, 108°24′E), Shaanxi Province, China. The trees were irrigated once a week with half-strength Hoagland’s nutrient solution [30]. Standard horticultural practices were followed. Inoculum preparation A monospore culture of D. mali originated from a diseased apple leaf with Marssonina blotch symptoms which was collected from the research orchard at Northwest A and F University. This isolate was cultured on a potato and carrot dextrose agar (PCDA: 10 g of fresh potato ‘Kexin No. 1’, 10 g of fresh carrot ‘Touxin-hong’, 2 g of dextrose, 0.7 g of agar, and 100 mL of distilled water) [31]. It was held on this medium for 30 d at 25°C under darkness. After incubation, fungal colonies from one Petri dish were scraped and ground with a mortar and pestle in 2 mL of sterile water to make culture suspensions. Excised, healthy ‘Fuji’ leaves were sterilized before their undersides were smeared with the suspensions. For test purposes, we chose ‘Fuji’ tissue for inoculum propagation because it was shown to be susceptible in our previous study (unpublished data). After smearing, the leaf tissues were placed in a plastic box, covered with plastic film, and kept at 25°C under darkness for 7 – 10 days. Sterile water was sprayed into the box twice each day to retain 90% humid conditions. When sporulation appeared on the infected leaves, the fungal inoculum was obtained by

removing the spores with sterile water. The inoculum concentration was adjusted to 1 9 106 conidia per milliliter. Treatments Experiments were conducted from September to October in 2011. Trees of uniform size were selected to determine the proper concentration of melatonin that would effectively protect them against infection. They were irrigated with 0, 0.05, 0.1, or 0.5 mM concentrations of melatonin solution. Each treatment group contained three replicates of ten plants. After 3 days of precultivation, each replicate was randomly divided in half. For one part, conidial suspensions (106 conidia mL
1) were brushed on both sides of the mature leaves (five per plant). The other part was inoculated with only sterile water as the uninfected controls. A misting system was used to maintain relative humidity above 90%. At 20 days postinoculation (dpi), the severity of infection for each leaf was scored on a scale of 0 – 5, where 0 = no disease symptoms, 1 = 1 – 10%, 2 = 11 – 30%, 3 = 31 – 50%, 4 = >50% of the leaf area showing lesions, and 5 = leaf fall. The formula for disease index was as follows: Disease index ¼ Sum of ðDisease class  Number of leaves in that classÞ  100 Total number of leaves  5 Based on preliminary results, we selected 0.1 mM melatonin for further study with 120 uniformly sized plants. Half were pretreated with 0.1 mM melatonin. The others received no pretreatment but only well-watered are labeled as nonmelatonin. After 3 days of precultivation, each group of nonmelatonin and melatonin-treated plants was then randomly divided in half to produce four new experimental groups. Two groups were inoculated on both sides of their mature leaves (five per plant) with D. mali suspension (106 conidia mL
1) (nonmelatonin/infected or melatonin/infected). The other two groups received only sterile water as the noninoculated controls (nonmelatonin/uninfected or melatonin/uninfected). Each group contained five replicates of six plants. All were then incubated in a greenhouse, where the relative humidity was maintained above 80% with a misting system. At Days 0, 2, 4, 6, 10, 16, and 20, leaves were sampled from plants in each treatment. All tissues were rapidly frozen in liquid nitrogen and stored at
80°C. Measurements of Fv/Fm, chlorophyll, and H2O2 Chlorophyll fluorescence was measured with an integrating fluorescence fluorometer (LI-6400-40 leaf chamber fluorometer; LICOR). After samples were dark adapted for 1 hr, the minimum fluorescence (F0) was measured under weak modulated irradiation (7000 lmol/m2/s) was applied to determine the maximum Chl fluorescence yield (Fm); Fv ⁄ Fm was calculated as (Fm –F0) ⁄ Fm. Chlorophyll was extracted with 80% acetone, and the content was determined spectrophotometrically according to the method of Lichtenthaler and Wellburn [32]. 427

Yin et al. Leaf tissues (0.1 g) from all treatment groups were ground in liquid nitrogen with a mortar and pestle and extracted with 2 mL of 5% (w/v) trichloroacetic acid, then centrifuged at 16,000 g for 10 min. The supernatant was used for the assay of H2O2, as described by Patterson et al. [33]. Extraction and assays of antioxidant enzymes Leaf samples (0.1 g) were ground in a chilled mortar with 1% (w/v) polyvinylpolypyrrolidone, then homogenized with 1.2 mL of 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA-Na2 and 0.3% Triton X-100. For the APX assay only, 1 mM ascorbate was added to this mixture. Each homogenate was centrifuged at 13,000 g for 20 min at 4°C and the supernatant was used for the following assays. CAT activity was determined by monitoring the decrease in absorbance at 240 nm due to decomposition of H2O2 (extinction coefficient of 39.4/mM/cm) [34]. APX activity was determined by monitoring the decrease in absorbance at 290 nm as reduced ASC was oxidized (extinction coefficient of 2.8/mM/cm) [35]. Peroxidase activity (POD) was assayed by monitoring the increase in absorbance at 470 nm due to guaiacol oxidation (extinction coefficient of 26.8/mM/cm) [36]. Extraction and assays of disease-related enzymes Phenylalanine ammonia-lyase activity was assayed spectrophotometrically following the method of Khan and Vaidyanathan [37]. Leaf samples (0.1 g) were homogenized with 5% polyvinylpolypyrrolidone and 2 mL of 100 mM Tris-HCl buffer (pH 7.5) containing 14 mM b-mercaptoethanol, 5 mM dithiothreitol, 10% Triton X-100, and 1% BSA in a chilled mortar. Afterward, the 1 mL supernatant was applied to a PD10 column equilibrated with 100 mM Tris-HCl (pH 7.5) buffer. Protein was eluted with 2 mL of elution buffer. The enzyme extraction was used for immediate analysis. PAL activity was determined spectrophotometrically by measuring the amount of trans-cinnamic acid formed at 290 nm (extinction coefficient of 17.4/mM/ cm). A reaction mixture that lacked L-phenylalanine was used as the control. Leaf samples (0.1 g) were homogenized with a chilled mortar and pestle in 2 mL of 100 mM sodium citrate buffer (pH 5.0) containing 1 mM EDTA and 5 mM b-mercaptoethanol. The homogenate was centrifuged for 10 min at 10,000 g. Afterward, 1 mL of the supernatant was de-salted on a PD10 column, using 100 mM sodium citrate buffer (pH 5.0) for elution. The first 2 mL after the void volume was collected and used for assaying chitinase and b-1,3-glucanase. Chitinase activity was measured as the release of N-acetyl-D-glucosamine from colloidal chitin [38]. The colorimetric determination of N-acetyl-D-glucosamine was conducted according to the method of Reissig et al. [39]. Inactivation of the enzyme served as the control. One unit of chitinase activity was expressed as the production of 1 nmol of N-acetyl-D-glucosamine per second at 45°C. Colloidal chitin was prepared according to the technique 428

described by Roberts and Selitrennikoff [40], using shrimp shell chitin powder (C9752; Sigma Chemical Co., Saint Louis, MO, USA). The activity of b-1,3-glucanase was determined by monitoring the amount of glucose released from laminarin (L9634; Sigma) [41]. Enzyme inactivation was the control. One unit of b-1,3-glucanase was represented by the production of 1 nmol of glucose-equivalent per second at 50°C. RNA isolation and quantitative real-time RT-PCR We evaluated gene expression via qRT-PCR. Complete coding sequences for the chitinase (FJ422811) and b-1,3glucanase (FJ598140) genes were obtained from http:// www.ncbi.nlm.nih.gov/. Their details are given in Table 1. Total RNA was extracted from leaves according to the method described by Chang et al. [42]. Poly(A)+ RNA was purified with a poly(A)+ Ttractâ mRNA Isolation Systems III kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. To remove any contaminating genomic DNA prior to cDNA synthesis, we treated the RNA with RNase-free DNase I (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA was quantified on a NanoDropTM 1000 spectrophotometer before and after DNase I treatment, and its quality and integrity were checked by electrophoresis through agarose gels stained with ethidium bromide. qRTPCR was performed using a PrimeScriptTMRT Reagent Kit (Takara, Kyoto, Japan) with oligo (dT)20 and random primers for cDNA synthesis, according to the manufacturer’s protocol. The amplified PCR products were quantified with an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) and a SYBR Premix Ex Taq kit (Takara). Transcripts of the Malus elongation factor 1 alpha gene (EF-1a; DQ341381) were used to standardize the cDNA samples for different genes. These qRT-PCR experiments were repeated three times, based on three separate RNA extracts from three samples. Statistical analysis All data were analyzed by one-way ANOVA, followed by Duncan’s multiple range tests. A P-value < 0.05 indicated

Table 1. Sequences for primers used in quantitative real-time RT-PCR Gene

Accession No.

Chitinase

FJ422811

b-1,3-glucanase

FJ598140

EF-1a

DQ341381

Primer Sequence (5′–3′) F: TGGAGGATGGGA AAGTGC R: GGGTGAGTTGGA TGGGTC F: TGCCGTAGGAAAC GAAAT R: TGATGGAGGAAAG GAATT F: ATTCAAGTATGCCT GGGTGC R: CAGTCAGCCTGTG ATGTTCC

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(D) Fig. 1. Effect of melatonin concentration on apple leaf phenotype at 20 dpi. Fully mature leaves were collected from plants pretreated with water only and no exogenous melatonin (A); 0.05 mM melatonin (B); 0.1 mM melatonin (C); or 0.5 mM melatonin (D).

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Fig. 2. Effect of melatonin concentration on disease index at 20 dpi for plants pretreated with 0, 0.05, 0.1, or 0.5 mM melatonin. Data represent means  S.D. of five replicate samples. *, significant difference between plants pretreated melatonin and nonmelatonin at P < 0.05 based on Duncan’s multiple range test.

a significant difference, and data were presented as the mean  standard deviation of five replicate samples.

Results Pretreatment with exogenous melatonin improved resistance to Diplocarpon mali by our apple plants (Fig. 1). After 20 days, leaves of inoculation-treated plants without melatonin application turned yellow and their leaf spots coalesced. The addition of 0.05 mM melatonin, 0.1 mM melatonin, or 0.5 mM melatonin alleviated the blotch damage to varying degrees. Compared with plants that were not pretreated, those in the 0.1 mM melatonin subgroup showed the greatest decline (54%) in the disease index, followed in second by a 51% decrease in the 0.5 mM melatonin subgroup plants (Fig. 2).

Fig. 3. Effect of melatonin concentration on maximum potential efficiency of Photosystem II (A) and chlorophyll content (B) in leaves at 20 dpi for plants pretreated with 0, 0.05, 0.1, or 0.5 mM melatonin. Data represent means  S.D. of five replicate samples. *,significant difference between inoculation and control at P < 0.05 based on Duncan’s multiple range test.

Infection by D. mali diminished the maximum potential efficiency of Photosystem II (PSII) by 15%, but that decrease was alleviated by melatonin (Fig. 3A), especially at concentrations of either 0.1 or 0.5 mM. Chlorophyll metabolism was also influenced by the blotch disease. For example, at Day 20, the total chlorophyll content from 429

Yin et al. infected plants was 78.24% of that measured in the nonmelatonin uninfected plants, whereas 0.1 mM melatonin or 0.5 mM melatonin subgroup plants had chlorophyll contents that were 99.10% or 98.14%, respectively, of that measured in the uninfected plants (Fig. 3B). Compared with the nonmelatonin/uninfected control plants, those in the nonmelatonin/infected group (i.e., without pretreatment) had significantly lower H2O2 contents at 4, 6, and 10 dpi (Fig. 4). The melatonin/infected group (addition of melatonin) had no significant changes on that parameter compared with melatonin/uninfected control. Activities of antioxidant enzymes involved in scavenging H2O2 during the infection period are shown in Fig. 5. In the nonmelatonin/infected group, CAT activity was 25% lower than in the nonmelatonin/uninfected control at 10 days and that low level was maintained (Fig. 5A). Application with 0.1 mM melatonin noticeably offset this decline in CAT activity compared with the melatonin/uninfected control (Fig. 5B). The APX activity for infected plants increased rapidly after 10 dpi when compared with the nonmelatonin/uninfected group (Fig. 5C). Exogenously applied melatonin had no significant impact on APX activity compared with the melatonin/uninfected group, except at 2 and 16 dpi (Fig. 5D). Compared with the nonmelatonin/infected control, POD activities within all pretreated subgroups increased during the plant interaction with D. mali (Fig. 5E, F). This was especially true at the beginning of disease exposure, indicating that pretreatment with 0.1 mM melatonin enhanced POD activity and then maintained it at a high level throughout the stress period. PAL activities in the leaves were first increased before gradually decreasing during the plant–D. mali interaction (Fig. 6A, B). Pretreatment with melatonin led to 67.28% greater activity compared with the nonmelatonin plants at Day 0. In the nonmelatonin sub-groups, chitinase activity did not differ significantly between infected and uninfected plants, except at Day 20 (Fig. 6C). When 0.1 mM melatonin was applied, the chitinase level increased significantly in infected plants at 6 dpi compared with the uninfected control, where it was maintained throughout the rest of the treatment period (Fig. 6D). In particular, applying 0.1 mM melatonin, the chitinase activity rose 150% at Day 0 compared with nonmelatonin plants. Compared with the uninfected controls, b-1,3-glucanase activities were dramatically elevated during the plant

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–fungi interaction (Fig. 6E, F). The average increase over the control occurred much earlier in groups that had received melatonin (Day 6) than in those that were not pretreated (Day 10). On Day 0, b-1,3-glucanse activity was 25% higher in plants within the 0.1 mM melatonin pretreatment subgroup than in the nonmelatonin group. Inoculation with D. mali led to the up-regulation of transcripts for both the chitinase and the b-1,3-glucanase genes (Fig. 7). At Day 0, the addition of melatonin was associated with a marked rise in the up-regulation of both. Relative expression in plants of the pretreated groups was 1.8- and 2.5-fold greater for chitinase and b-1,3-glucanase, respectively, than in those from the nonmelatonin subgroups.

Discussion Marssonina apple blotch is one of the most severe apple diseases in production regions. Our results demonstrated that exogenous melatonin clearly had a role in protecting trees from infection, reducing the number of lesions and slowing their rate of expansion. Moreover, for infected plants, melatonin pretreatment helped maintain the high potential efficiency of Photosystem II and improved the total chlorophyll content when leaves were compared with nonmelatonin tissues. Such chlorophyll-preserving effects of melatonin have also been reported in apple leaves during senescence [25, 26] or when under salinity-induced stress [24], as well as in drought-stressed cucumber seedlings [23]. This melatonin influence exhibits a dose-dependent relationship [19, 23, 43]. Consistent with those earlier conclusions, we found that 0.1 mM melatonin was most effective in protecting against D. mali. That same concentration is also best at reversing PEG-induced inhibition of germination by cucumber seeds [23] and can also stimulate lateral and adventitious root formation in Arabidopsis seedlings [43]. However, our laboratory is the first to report that exogenous melatonin has a mitigating effect against pathogen infection. H2O2 is considered the most versatile and stable ROS, with several possible functions in a plant’s defense strategies [44]. In addition to its direct antimicrobial effects, such as inhibiting the germination of spores of many fungal pathogens, H2O2 is involved in the oxidative crosslinking of cell wall glycoproteins [45]. It also has a key role

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Fig. 4. Effect of melatonin on leaf H2O2 content after inoculation. Leaves were pretreated with water only and no exogenous melatonin (A) or 0.1 mM melatonin (B). Data represent means  S.D. of five replicate samples.*, significant difference between inoculation and control at P < 0.05 based on Duncan’s multiple range test.

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Melatonin improves disease resistance

Fig. 5. Effect of melatonin on antioxidative enzymes after inoculation. CAT activities in leaves without exogenous melatonin (A) or pretreated with 0.1 mM melatonin (B); APX activities in leaves without exogenous melatonin (C) or pretreated with 0.1 mM melatonin (D); POD activities in leaves without exogenous melatonin (E) or pretreated with 0.1 mM melatonin (F). Data represent means  S.D. of five replicate samples. *, significant difference between inoculation and control at P < 0.05 based on Duncan’s multiple range test.

Fig. 6. Effect of melatonin on enzymes related to disease resistance after inoculation. PAL activities in leaves without exogenous melatonin (A) or pretreated with 0.1 mM melatonin (B); Chitinase activities in leaves without exogenous melatonin (C) or pretreated with 0.1 mM melatonin (D); Glucanase activities in leaves without exogenous melatonin (E) or pretreated with 0.1 mM melatonin (F). Data represent means  S.D. of five replicate samples. *, significant difference between inoculation and control at P < 0.05 based on Duncan’s multiple range test.

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in orchestrating the hypersensitive cell death response during the expression of plant disease resistance [13]. As a component of basal resistance, some successful pathogens

can suppress ROS production [46]. In our study, inoculation with D. mali was associated with a significant decrease in H2O2 contents at 4, 6, and 10 dpi. However, 431

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no significant changes were found when we compared between melatonin/infected plants and melatonin/uninfected plants. This meant that melatonin relatively enhanced ROS production over that measured in the nonmelatonin/infected tissues. By contrast, under abiotic stresses such as high salinity or drought, melatonin treatment is correlated with reduced ROS production [23, 24]. This perhaps because of a link with the amounts of ROS accumulated. Consequently, under either abiotic or biotic stress, melatonin may modulate ROS levels by maintaining steady-state intracellular H2O2 concentrations. CAT and APX are important scavenging enzymes that remove H2O2 through a mechanism known as the Halliwell–Asada–Foyer pathway [47]. When our plants were inoculated with D. mali, CAT levels decreased and APX increased beginning from 10 dpi. Melatonin pretreatment had little effect on those activities when compared with the melatonin/uninfected control. These results are consistent with the pattern found for H2O2 production, which indicated that pathogen infection did not influence the oxidation-reduction system within pretreated plants. This also suggested that melatonin plays a critical role in maintaining the oxidation-reduction system at steadystate levels. Peroxidases form a complex family of proteins that catalyze the oxido-reduction of various substrates using H2O2 [48]. Peroxidases participate in wall-building processes, for example, oxidation of phenols, suberization, and lignification of host cells during the defence reaction against pathogenic agents [49]. Thus, they are an important component in the plant-response system to fungal attack. We showed here that POD activity was significantly elevated at the beginning of the inoculation period, indicating a link between this activity and exposure to 0.1 mM melatonin. Furthermore, this activity remained at a high level throughout the experiment. Such modifications may improve the effectiveness of cell walls to act as a barrier that slows the spread of a pathogen. Enhanced POD activity has been correlated with disease resistance in rice [50], 432

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Fig. 7. Effect of melatonin on transcript abundance of chitinase and b-1,3glucanase genes after inoculation. Total RNA was isolated from samples taken at different time points, converted to cDNA, and subjected to real-time RTPCR. Expression levels were calculated relative to expression of Malus EF-1a mRNA. Relative expression of chitinase gene in leaves without exogenous melatonin (A) or when pretreated with melatonin (B); Relative 0.1 mM expression of b-1,3-glucanase gene in leaves without exogenous melatonin (C) or when pretreated with 0.1 mM melatonin. Data represent means  S.D. of three replicate samples.

wheat [49], and tobacco [51]. Furthermore, antisense expression of peroxidase in Arabidopsis makes plants more susceptible to a broad range of fungal and bacterial pathogens [52]. Phenols and polyphenols are widely distributed among higher plants and are an important player in defence responses. PAL is the key enzyme of the phenylpropanoid pathway, which leads to the synthesis of defence-related compounds [53]. In the Arabidopsis–Peronospora pathosystem, PAL is involved in synthesizing salicylic acid (SA) and precursors for lignification [54]. In fact, systemic acquired resistance (SAR) requires SA [55]. The lignification of cucumber epidermal cell walls directly inhibits the penetration of Colletotrichum lagenarium [56]. After infection by the virulent fungal pathogen Cercospora nicotianae, transgenic tobacco plants with suppressed PAL levels exhibit more rapid development and expansion of lesions than do wild-type plants [57]. We also determined here that PAL was a resistance component in the pathosystem of apple and D. mali. Its activity was significantly increased and then maintained at a high level in pretreated tissues when compared with nonmelatonin control plants. This suggested that melatonin might regulate the phenylpropanoid pathway to enhance the resistance of apple trees against D. mali. As pathogenesis-related proteins, chitinase and b-1,3glucanase can protect plants against fungal infection in two ways. First, these proteins can directly weaken and decompose the fungal cell walls [15]. Second, oligosaccharide elicitors, released through those digested walls, can induce a consequent chain of defense reactions [58]. Activities by both chitinase and b-1,3-glucanase can be significantly induced by infection, as shown in several pathosystems [38, 59]. Additionally, the activities of those are higher during an incompatible interaction than in one that is compatible [60]. For example, when pea pods are infected with Fusarium solani f. sp. phaseoli, chitinase and b-1,3-glucanse activity is enhanced by 9- and 4-fold, respectively [61]. Induction of the wheat b-1,3-glucanse

Melatonin improves disease resistance gene transcript is more than 60-fold higher in the resistant ‘Shannong0431’ line than in susceptible ‘Wenmai6’ after infection with Rhizoctonia cerealis [62]. We also noted that, for pretreated leaves, activities and transcript induction of chitinase and b-1,3-glucanse were significantly raised when inoculation began, and they remained at high levels throughout the experimental period. Our results indicated that melatonin could improve and maintain these activities, thereby possibly contributing to greater disease resistance in those apple plants. In conclusion, the application of exogenous melatonin to apple trees prior to inoculation with D. mali alleviates disease damage by reducing the number of lesions and inhibiting their expansion. This in turn enables plants to maintain a high potential efficiency of Photosystem II, retain their normal chlorophyll contents, keep intracellular H2O2 concentrations at steady-state levels and increase the activities of plant defence-related enzymes. All of these factors are probably the main reasons for why melatonin apparently enhances disease resistance. Therefore, this pretreatment approach represents a promising strategy for protecting apple trees against the ill effects of D. mali infection. Employing this tool can also greatly reduce environmental pollution that is generally associated with chemical pesticide applications. Ours is the first report on the protective role of melatonin against pathogens. Nevertheless, the mechanism by which it assists in the acquisition of resistance requires further exploration.

Acknowledgements This study was supported by the earmarked fund from the China Agriculture Research System. The authors are grateful to Priscilla Licht for help in revising our English composition.

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