β-Aminobutyric acid induces resistance of mango fruit to postharvest anthracnose caused by Colletotrichum gloeosporioides and enhances activity of fruit defense mechanisms

Scientia Horticulturae 160 (2013) 78–84

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␤-Aminobutyric acid induces resistance of mango fruit to postharvest anthracnose caused by Colletotrichum gloeosporioides and enhances activity of fruit defense mechanisms Zhengke Zhang a,c , Dongping Yang b , Bo Yang b , Zhaoyin Gao a , Min Li a , Yueming Jiang c , Meijiao Hu a,∗ a

Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China College of Environment and Plant Protection, Hainan University, Haikou 570228, PR China c South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, PR China b

a r t i c l e

i n f o

Article history: Received 18 March 2013 Received in revised form 14 April 2013 Accepted 17 May 2013 Keywords: Mango Anthracnose ␤-Aminobutyric acid BABA Colletotrichum gloeosporioides Induced resistance

a b s t r a c t The effect of ␤-aminobutyric acid (BABA) on control of anthracnose caused by Colletotrichum gloeosporioides in mango fruit and its possible mechanisms were investigated. The results show that BABA treatments effectively suppressed the expansion of lesion in mango fruit inoculated with C. gloeosporioides during storage at 25 ◦ C, with the greatest efficacy being obtained using 100 mM BABA. However, BABA at 25–400 mM did not exhibit direct antifungal activity against C. gloeosporioides in vitro. Furthermore, BABA treatment at 100 mM enhanced the activities of ␤-1,3-glucanase (GLU), chitinase (CHT) and phenylalanine ammonia lyase (PAL). BABA treatment also contributed to the accumulation of hydrogen peroxide (H2 O2 ), while decreasing the rate of superoxide radical (O2 • − ) production. Concurrently, BABA increased the activity of superoxide dismutase (SOD), while inhibiting catalase (CAT) and ascorbate peroxidase (APX) activities. These results indicate that increased disease resistance of mango fruit after BABA treatment during storage might be attributed to an elicitation of defense response involving in the enhancement of defense-related enzyme activities and modulation of antioxidant system activities. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Mango (Mangifera indica L.) is considered an important fruit crop grown throughout the tropics because of its favorable flavor, rich nutrition and high marketing value. However, mango is highly susceptible to postharvest decay caused by various pathogens, leading to major economic losses (Tian et al., 2010). Anthracnose caused by Colletotrichum gloeosporioides (Penz) is well known as one of the most destructive postharvest diseases in mango fruit. The pathogen can attack immature mango fruit as a latent infection, and the lesions progressively appear after storage (Dodd et al., 1989). Disease control is achieved primarily by postharvest application of fungicides such as benomyl and prochloraz, alone or in combination with other treatments (Johnson et al., 1997). However, because of problems related to fungicide toxicity, development of fungicide resistance by pathogens and potential adverse effects on the environment and human health, alternative strategies for controlling postharvest rot have been proposed (Terry and Joyce, 2004; Droby et al., 2009).

∗ Corresponding author. Tel.: +86 898 66969242; fax: +86 898 66969242. E-mail addresses: [email protected], [email protected] (M. Hu). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.05.023

Induction of resistance to pathogens in harvested crops using physical, biological, and chemical elicitors is becoming a promising approach for controlling postharvest diseases as an alternative to fungicides (Terry and Joyce, 2004). It has been previously reported that several resistance-inducing chemicals including salicylic acid (SA), isonicotinic acid (INA), benzo (1,2,3) thiadiazole7-carbothionic acid S-methyl ester (BTH) and their analogs under low concentrations effectively provide the protection against challenge infection of C. gloeosporioides to ‘Keitt’ mango seedling, leaves and fruit (Santiago et al., 2006), and the authors also suggest that other chemical elicitors should be evaluated to clarify the induced resistance in mango tissues. The non-protein amino acid ␤-aminobutyric acid (BABA), a rare compound found in nature, has been shown to induce resistance against a broad range of pathogenic organisms, including fungi, bacteria, viruses and nematodes in plants (Zimmerli et al., 2001; Cohen, 2002; Van der Ent et al., 2009; Quaglia et al., 2011). The resistance is not based on direct defense activation by the BABA, but on faster and stronger activation of inducible defense reaction once the plant is exposed to the pathogen (Zimmerli et al., 2000, 2001; Ton et al., 2005). The phenomenon is referred to ‘priming’ which prompts plant to an alarmed state of defense (Conrath et al., 2006). The mechanisms of BABA-induced resistance to pathogens

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through priming of defense response have been intensively studied in Arabidopsis (Zimmerli et al., 2000, 2001; Ton and Mauch-Mani, 2004; Ton et al., 2005; Van der Ent et al., 2009) and have also been partially elucidated in several harvested crops. For instance, BABA treatment restricts lesion expansion in stored grapefruit and apple fruit inoculated with Penicillium digitatum and P. expansum, which is associated with the induction of an increase in defense-related enzyme activities and their gene expression (Porat et al., 2003; Zhang et al., 2011). Yin et al. (2010) found that several antifungal metabolites, including phenols, flavonoids and lignin related to the defense response are enhanced by BABA treatment, thereby contributing to the establishment of systemic resistance in potato tuber during storage. These findings indicate that BABA might be a promising priming agent to induce effective resistance against postharvest disease. However, to the best of our knowledge, no information is available on the inhibitory effect of BABA against C. gloeosporioides and its possible mechanism involved in induced resistance in mango fruit during storage under ambient temperature. The objectives of this study were to investigate the effects of postharvest treatment with BABA on the inhibition of anthracnose caused by C. gloeosporioides in mango fruit during storage at 25 ◦ C, to evaluate antifungal activity of BABA against C. gloeosporioides in vitro, as well as evaluate the effects of BABA on defenserelated enzymes (phenylalanine ammonia lyase, ␤-1,3-glucanase and chitinase) and reactive oxygen species (ROS) metabolism (superoxide radical, hydrogen peroxide, superoxide dismutase, catalase and ascorbate peroxidase), and then to understand the role of BABA in inducing disease resistance in mango fruit. 2. Materials and methods 2.1. Plant material Mature green mango (M. indica L. cv. Guifei) fruit at 126.3 ± 5.6 N of firmness and 8.5 ± 0.2 ◦ Brix of total soluble solids (n = 10) were harvested from a commercial orchard located in Dongfang city, Hainan Province of China. Fruit were transported to the postharvest laboratory within 6 h. Fruit of uniform size and appearance, and that are free of visible symptoms of any disease were selected for the experiments.

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growth as expressed by diameter (mm) was recorded after 1, 3, 5 and 7 days of incubation at 25 ± 1 ◦ C. Each treatment concentration was replicated three times, and the experiment was repeated twice. The effects of BABA on spore germination of C. gloeosporioides were assayed in PDB using the method of Yao and Tian (2005). Aliquots of 100 ␮L of spore suspension at 1 × 106 were individually transferred to glass tubes with PDB, which contained BABA at different concentrations (0, 25, 50, 100, 200 and 400 mM). All the tubes were put on a rotary shaker at 100 rpm at 25 ◦ C and incubated for up to 12 h. Approximately 200 spores were measured at 3 h intervals for germination rate per replicate, with 3 replicates for each treatment. The experiment was repeated twice. 2.4. BABA treatment and inoculation Mango fruit were disinfected with 2% (v/v) sodium hypochlorite for 2 min, rinsed with tap water, air-dried and then divided randomly into 4 treatment groups, 120 fruit for each group. Three groups were dipped in 25, 50 and 100 mM BABA solutions containing 0.05% (v/v) Tween 80 at 25 ± 1 ◦ C for 5 min as described in Yin et al. (2010). Another group (control) was treated with distilled water containing 0.05% (v/v) Tween 80 for 5 min. After 24 h of treatments with BABA or water, a uniform wound (3 mm deep × 3 mm wide) was made at the equator of each fruit using a sterile dissecting needle, followed by inoculation of a 10-␮L conidial suspension of C. gloeosporioides (1 × 106 spores mL−1 ) into each wounded site. Inoculated fruit were placed in covered plastic boxes with small holes and stored at 25 ± 1 ◦ C and RH 85–90%. Diameters of lesions caused by C. gloeosporioides in the mango fruit were recorded at 4 and 6 days after inoculation, each treatment had three replicates with 15 fruit per replicate. The remaining fruit with inoculation were used for determination of physio-biochemical parameters as described below. 2.5. Sample collecting The fruit mesocarp tissue at 3–10 mm below the skin and 5–15 mm from the edge of the inoculated lesion was daily taken with a stainless steel cork borer during storage. The tissue samples were frozen in liquid nitrogen and stored at −80 ◦ C until analyzed. 2.6. Enzyme assays

2.2. Pathogen C. gloeosporioides was isolated from infected mango fruit and kept on potato dextrose agar (PDA) at 4 ◦ C. The pathogen was inoculated into mango fruit wounds and re-isolated into potato dextrose broth (PDB) for further use. The spores of the pathogen were removed from 2-week-old PDB cultures incubated at 25 ◦ C and then suspended in 10 mL of sterile distilled water containing 0.05% (v/v) Tween 80. The suspension was filtered through 4 layers of sterile cheese cloth to remove the mycelia. The number of spores was counted with a hemacytometer, and the concentration was adjusted to 1 × 106 spores mL−1 with sterile distilled water. 2.3. In vitro antifungal activity The effects of BABA (Yuanye Co., Ltd., Shanghai, China) on the in vitro growth of C. gloeosporioides were tested. BABA solution mixed with molten PDA to give a total volume of 20 mL per petri plate (diameter: 120 mm). BABA concentrations in the PDA were 0, 25, 50, 100, 200 and 400 mM. After the PDA had solidified, an 8-mm diameter disk of mycelial mat from a 1-week-old culture was placed in the center of each Petri plate containing PDA and BABA. The mycelial

The fruit treated with BABA at the optimum concentration of 100 mM, based on the results of antifungal activity in vivo, were used for analysis of enzymes and metabolites. Five-gram samples of frozen mango tissue derived from three fruit, with 3 replicates for each treatment, were ground with a mortar and pestle on ice and homogenized with various pre-cooled extracting buffers as follows: 5 mL of 100 mM boric acid buffer (pH 8.8) containing 4% (w/v) polyvinylpyrrolidone, 1 mM ethylene diamine tetraacetic acid (EDTA) and 50 mM ␤-mercaptoethanol for phenylalanine ammonia lyase (PAL); 5 mL of 50 mM sodium acetate buffer (pH 5.2) containing 1 mM EDTA, 5 mM ␤-mercaptoethanol and 5 mM ascorbic acid for ␤-1,3-glucanase (GLU) and chitinase (CHT); 5 mL of 100 mM sodium phosphate buffer (pH 7.5) containing 5% (w/v) polyvinylpyrrolidone and 5 mM dithiothreitol for superoxide dismutase (SOD) and catalase (CAT), and 5 mL of 100 mM potassium phosphate buffer (pH 7.5) containing 2% polyvinylpyrrolidone, 1 mM ascorbic acid and 1 mM EDTA for ascorbate peroxidase (APX). The extracts were then centrifuged at 15,000 × g for 20 min at 4 ◦ C. The supernatants were used for the enzyme assays. PAL activity was assayed as described by method of Assis et al. (2001), with some modifications. Enzyme extract (0.5 mL)

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was incubated with 0.5 mL of 20 mM l-phenylalanine and 2 mL of 50 mM borate buffer (pH 8.8) at 37 ◦ C for 1 h. The reaction was stopped with 0.1 mL HCl (6 M). The increase in absorbance at 290 nm due to the formation of trans-cinnamate was measured spectrophotometrically. PAL activity was expressed as U290 , where U290 = 0.01 A290 mg−1 protein h−1 . GLU activity was assayed by measuring the amount of reducing sugar released from the substrate according to the method of Ippolito et al. (2000), with some modifications. Enzyme extract was dialyzed at 4 ◦ C for 12 h and then centrifuged at 12,000 × g for 20 min at 4 ◦ C. The supernatant (2 mL) was incubated with 0.2 mL of laminarin (0.5%, w/v) for 60 min at 37 ◦ C. Afterward, 1 mL of the mixture was removed and diluted 1:1 with sterile distilled water. The color reaction was terminated by adding 1.5 mL of 3,5dinitrosalicylate and boiling for 5 min in a water bath. The solution was diluted to 25 mL with distilled water, and the amount of reducing sugar was measured spectrophotometrically at 540 nm. Activity values were expressed as U ␮g−1 protein. One unit (U) was defined as the enzyme activity catalyzing the formation of 1 nmol s−1 glucose equivalent. CHT activity was assayed using the method of Wirth and Wolf (1990), with slight modification. CHT activity was measured by mixing 1.5 mL of a crude enzyme dialytic solution as described for GLU, with 2 mL of 2% dye-labeled carboxymethylchitin in 50 mM sodium acetate buffer (pH 5.0). After incubation at 37 ◦ C for 1 h, the reaction was stopped by adding 0.1 mL of 1.0 M HCl. The mixture was cooled on ice and centrifuged at 15,000 × g for 5 min. The absorbance of the supernatant was measured at 550 nm. CHT activity was calculated and expressed in U ␮g−1 protein. One unit (U) was defined as the amount of enzyme required to catalyze the formation of 1 nmol min−1 of product (N-acetyl-d-glucosamine). SOD activity was determined photochemically as described in Toivonen and Sweeney (1998). The reaction solution contained 50 mM sodium phosphate (pH 7.8), 13 mM methionine, 100 ␮M EDTA and 75 ␮M nitro-blue-tetrazolium (NBT). To 2.7 mL of this solution were added 0.1 mL of enzyme extract and 0.2 mL of 200 ␮M riboflavin. The tubes were then placed in a 40 W-fluorescent-light incubator for 15 min. Blue formazan formation was then monitored by recording the absorbance at 560 nm. One unit (U) of SOD activity is defined as the amount of enzyme that causes a 50% inhibition of NBT reduction under assay conditions. The results are reported as U mg−1 protein. CAT activity was assayed according to the method of Chance and Maehly (1955). The reaction mixture consisted of 50 mM sodium phosphate (pH 7.0), 20 mM H2 O2 and 100 ␮L enzyme extract in a total volume of 3 mL. H2 O2 degradation was measured by the decrease in absorbance at 240 nm. CAT activity is expressed as U240 , where U240 = 0.01 A240 mg−1 protein min−1 . APX activity was assayed using the method of Nakano and Asada (1987), with some modifications. The mixture contained 50 mM potassium phosphate (pH 7.0), 0.25 mM ascorbic acid, 0.05 mM EDTA and 0.2 mL enzyme extract in a total volume of 2.7 mL. After adding 0.3 mL of H2 O2 to a final concentration of 0.2 mM, the change in absorbance was monitored at 290 nm. APX activity is expressed as U290 , in which U290 = 0.01A290 mg−1 protein min−1 . Protein content in the enzyme extracts was determined using the Bradford (1976) method with bovine serum albumin as the standard.

2.7. Assays of reactive oxygen determination The superoxide radical (O2 •− ) production rate was determined using the method of Elstner and Heupel (1976), with some modifications. Five gram samples of frozen mango tissue derived from

three fruit, with 3 replicates for each treatment, were ground with a mortar and pestle on ice, and homogenized with 5 mL of 50 mM sodium phosphate buffer (pH 7.8) containing 1 mM EDTA, 1% polyvinylpyrrolidone (PVP, w/v) and 0.3% Triton X-100. The homogenate was centrifuged at 5000 × g for 15 min at 4 ◦ C. A 0.5mL aliquot of the supernatant was mixed with 1.0 mL of 100 mM sodium phosphate buffer (pH 7.8) and 0.5 mL of 10 mM hydroxylamine hydrochloride. After incubation for 20 min at 25 ◦ C, 1 mL of the above reaction mixture was added to 1 mL of 17 mM 4aminobenzene sulfonic acid, and 1 mL of 7 mM ␣-naphthylamine was added to the mixture for a further 20 min at 25 ◦ C. Then, 4 mL of n-butanol was added into the reaction mixture. The absorbance of the n-butanol phase was measured at 530 nm used for the measurement of O2 •− The O2 •− production rate was expressed as ␮mol min−1 g−1 FW. The hydrogen peroxide (H2 O2 ) content was determined according to the method described by Prochazkova et al. (2001). Five-gram samples of frozen mango tissue derived from three fruit, with 3 replicates for each treatment were homogenized on ice with 5 mL sodium phosphate buffer (50 mM, pH 6.5). The homogenate was centrifuged at 12,000 × g for 15 min at 4 ◦ C. Thereafter, 1 mL of the extracted solution was mixed with 0.1 mL of 0.1% titanium tetrachloride (dissolved in concentrated hydrochloric acid, v/v) and 0.2 mL of concentrated ammonia solution, and centrifuged at 12,000 × g for 15 min at 4 ◦ C. The precipitate was washed repeatedly by cold acetone and dissolved in 3 mL of 2 M H2 SO4 . After centrifugation at 3000 × g for 10 min at 4 ◦ C, the absorbance of the supernatant was measured at 410 nm. The H2 O2 content was expressed as ␮mol g−1 FW. 2.8. Statistical analysis The data were subjected to one-way analysis of variance (ANOVA) using SAS statistical software (Version 8, SAS Institute, Cary, NC, USA). Fisher’s least significant differences (LSD, p = 0.05) were determined to compare differences between means following a significant ANOVA effect. All data are presented as the mean ± standard error of the mean (SEM). 3. Results 3.1. Effect of BABA treatment on the anthracnose lesion diameter in mango fruit As shown in Fig. 1, BABA treatment effectively reduced the lesion diameter of anthracnose caused by C. gloeosporioides during storage at 25 ◦ C, and the inhibitory effect was positively correlated with the concentration of BABA. Among the test concentrations, 100 mM BABA showed the strongest inhibitory efficacy, with average lesion diameters on days 4 through 6 after inoculation was averagely 35, 34 and 24% lower than that of control, 25 mM and 50 mM BABAtreated fruit, respectively. 3.2. Effect of BABA on mycelial growth and spore germination of C. gloeosporioides in vitro The mycelial colony of C. gloeosporioides in PDA medium without BABA rapidly grew from 8 to 70.4 mm diameter during 7 days of incubation period at 25 ◦ C. Adding BABA at concentrations of 25–400 mM in PDA medium did not significantly influence the mycelial growth of C. gloeosporioides in vitro (Fig. 2A). Similarly, spore germination C. gloeosporioides were not obviously different between BABA treatments and control during incubation of 12 h (Fig. 2B).

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Fig. 1. Effect of BABA on anthracnose lesion diameter in mango fruit inoculated with C. gloeosporioides during storage at 25 ◦ C. Bars represent standard errors of means of three replicate assays. Values labeled with different letters between control and BABA-treated fruit at each time point are significantly different using LSD test at P = 0.05.

Fig. 3. Effect of 100 mM BABA on GLU (A), CHT (B) and PAL (C) activities in mango fruit during storage at 25 ◦ C. Bars represent standard errors of the means of three replicate assays. Asterisk (*) represents that values are significantly different between control and BABA-treated fruit at the same time point using LSD test at P = 0.05.

3.3. Effect of BABA treatment on GLU, CHT and PAL activities in mango fruit

Fig. 2. Effect of BABA on mycelial growth (A) and spore germination (B) of C. gloeosporioides in vitro at 25 ◦ C. Bars represent standard errors of the means of three replicate assays. Asterisk (*) represents that values are significantly different between control and BABA-treated fruit at the same time point using LSD test at P = 0.05.

GLU and CHT activities in the control fruit steadily increased and reached their highest values at 4 and 5 days of storage, respectively, followed by a sharp decline (Fig. 3A and B). BABA treatment resulted in significant increases in both GLU and CHT activities compared to the control during storage (Fig. 3A and B). PAL activity gradually increased in the control fruit within the first 4 days of storage and then began to decrease (Fig. 3C). PAL activity in BABAtreated fruit exhibited a similar mode to that in control fruit, while

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Fig. 4. Effect of 100 mM BABA on the rate of O2 • − production (A) and H2 O2 content (B) in mango fruit during storage at 25 ◦ C. Bars represent standard errors of the means of three replicate assays. Asterisk (*) represents that values are significantly different between control and BABA-treated fruit at the same time point using LSD test at P = 0.05.

it was significantly higher than that of control fruit during storage (Fig. 3C). 3.4. Effect of BABA treatment on the O2 •− production rate and H2 O2 content in mango fruit The O2 •− roduction rate in control fruit increased from an initial value of 0.63 to a maximum of 0.87 ␮mol min−1 g−1 FW at 5 days of storage, followed by a steady decline as the fruit ripened (Fig. 4A). BABA treatment resulted in a significantly lower rate of O2 •− production, as the average value in BABA-treated fruit was 14% lower than that of control fruit through 2–7 days of storage (Fig. 4A). The H2 O2 content in control fruit showed a continuous increase during storage (Fig. 4B). The remarkable difference in H2 O2 level between control and BABA treatment began to appear after 2 days of storage, with the average content in BABA-treated fruit being 16% higher than that of control fruit within 2 to 7 days during storage (Fig. 4B).

Fig. 5. Effect of 100 mM BABA on SOD (A), CAT (B) and APX (C) activities in mango fruit during storage at 25 ◦ C. Bars represent standard errors of the means of three replicate assays. Asterisk (*) represents that values are significantly different between control and BABA-treated fruit at the same time point using LSD test at P = 0.05.

lower than those in control fruit throughout the storage period (Fig. 5C and D).

3.5. Effect of BABA treatment on SOD, CAT and APX activities in mango fruit

4. Discussion

The activity of SOD in control and BABA-treated fruit declined during storage, whereas SOD activity in the latter was higher than that in the former throughout the storage period (Fig. 5A). In general, the activities of CAT and APX in control fruit exhibited a gradual increase during storage (Fig. 5C and D). The activities of CAT and APX in fruit were markedly inhibited by BABA treatment, being much

The data presented here show that postharvest BABA treatment effectively suppressed the anthracnose caused by C. gloeosporioides in ‘Guifei’ mango fruit during storage at 25 ◦ C, with a relatively high concentration (100 mM) showing the strongest inhibitory effect (Fig. 1). Previous studies have shown that BABA as applied by foliar sprays, soil drench or dipping treatment induces local and systemic

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resistance against C. coccoides in pepper plants (Hong et al., 1999; Sang and Kim, 2011), and Colletotrichum orbiculare and C. lagenarium in cucumber plants (Jeun et al., 2007; Walz and Simon, 2009; Sang and Kim, 2011). These findings indicate that BABA may be very effective to induce resistance against a range of Colletotrichum species. In addition, the similar result for infection control due to BABA treatment has also been obtained in other harvested crops, including citrus (Porat et al., 2001, 2002), grapefruit (Porat et al., 2003), potato tuber (Yin et al., 2010) and apple fruit (Quaglia et al., 2011; Zhang et al., 2011). However, we found that BABA was ineffective in inhibiting the mycelial growth and spore germination of C. gloeosporioides in vitro, despite the use of a wide range of concentrations (25–400 mM) in our study (Fig. 2). Consistent with our results, BABA did not exhibit direct antifungal activity against Phytophthora infestans (Cohen, 1994), Alternaria alternata (Reuveni et al., 2003) or C. orbiculare (Lee et al., 2005). By contrast, numerous studies have confirmed the direct, dose-dependent antifungal activity of BABA against P. digitatum, P. italicum and P. expansum (Porat et al., 2003; Tavallali et al., 2008; Quaglia et al., 2011; Zhang et al., 2011), Fusarium sulphureum (Yin et al., 2010), Sclerotinia scleˇ sek rotiorum (Marcucci et al., 2010) and Leptosphaeria maculans (Saˇ et al., 2012). The inhibition mode of BABA in vitro growth of fungal pathogens is related to its stimulation for plasma membrane disintegration and leakage of intracellular protein and sugars out of the cell (Zhang et al., 2011). The above different findings suggest that multiple factors, including specific fungal species, fungal culture conditions and BABA concentrations may have contributed to variation of the antifungal effect of BABA in vitro. In a previous independent study, we demonstrate that postharvest dip treatments with BABA at 25–100 mM may decrease natural disease incidence by approximately 6–33%, but not affect the softening, ethylene production, respiration rate, total soluble solids content and total titratable acidity in ‘Guifei’ mango fruit during storage at 25 ◦ C (data not shown), indicating that the control of infection by BABA in mango fruit might be associated with the activation of defense mechanisms and rule out the possibility of interference by fruit ripening. The present results provide evidence that BABA strengthens the defense system in mango fruit by enhancing the activity of pathogenesis-related (PR) proteins, activating the phenylpropanoid pathway and modulating the ROS metabolism. GLU and CHT are two types of most characterized PR proteins, playing very important roles in the interactions between host and pathogen. GLU has been shown the direct hydrolysis for ␤-1,3linked glucans, which are major components of the cell walls of fungi, and may also aid indirectly in resistance through the release of oligosaccharides in plants. CHT plays a crucial role in degrading chitin, an essential component of fungal cell walls (Graham and Sticklen, 1994). GLU can act synergistically with CHT to inhibit fungal growth (Schneider and Ullrich, 1994; Kim and Hwang, 1997). A significant accumulation of GLU and CHT indicates an overall resistance against pathogens. PAL is a key regulatory enzyme in the phenylpropanoid pathway, and an increase in PAL activity is associated with biosynthesis of active metabolites such as phytoalexins, phenols, tannin, lignins and SA in plant defense pathways (Singh et al., 2010). In the current study, we found that the activities of defense-related enzymes (GLU, CHT and PAL) were enhanced by BABA treatment (Fig. 3), which might be responsible for inhibiting anthracnose in mango fruit. Similarly, BABA treatment increased the activities of GLU and CHT in ‘Golden Delicious’ apple fruit against blue mold caused by P. expansum (Zhang et al., 2011). It has been shown that BABA treatment enhances the PAL activity in grapefruit (Porat et al., 2003) and up-regulates the expression of the GLU and CHT genes in citrus fruit (Porat et al., 2001, 2002), thus providing protection from P. digitatum infection in both fruits. However, only minor accumulation of PR proteins was observed

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by western blot analysis in lettuce after leaves were sprayed with BABA (500–2000 ␮g mL−1 ), suggesting that BABA-mediated resistance of lettuce against downy mildew may have been through a SA-independent signaling pathway (Cohen et al., 2010). An oxidative burst, which is characterized by the rapid generation of ROS, mainly including O2 •− and H2 O2 , is considered one of the most prominent and earliest defense responses in the host-pathogen interaction, involving in either directly killing the pathogen or restricting its ingress due to cytotoxicity (Apel and Hirt, 2004). ROS also participate in other resistance mechanisms such as reinforcement of the plant cell wall (lignification, cross-linking of cell wall structural proteins), the hypersensitive response and phytoalexin synthesis (Małolepsza, 2006). However, excess H2 O2 production may lead to lipid peroxidation, a prominent feature of plant senescence (Torres et al., 2006). In the present study, BABA treatment caused an increase in H2 O2 production in mango fruit, whereas the rate of O2 •− production was maintained at a lower level compared with control fruit (Fig. 4). The higher H2 O2 level that resulted from BABA treatment might be an important element in the defense system and could play a role for halting the growth of pathogens in mango fruit. Such a rapid accumulation of H2 O2 relating to a strengthened host resistance against Bremia lactucae after BABA treatment has also been observed in the lettuce (Cohen et al., 2010). The contribution of ROS to the stimulation of disease resistance in fruit is generally controlled by an array of antioxidant enzymes, including SOD, CAT and APX. The data presented here show increased activity of SOD and decreased activities of CAT and APX after BABA treatment (Fig. 5). SOD catalyzes the disproportion of O2 •− to O2 and H2 O2 , which can be subsequently decomposed by CAT and APX (Mittler, 2002). Removal of these ROS is crucial in decreasing the risk of • OH production via metal-dependent Haber–Weiss or Fenton reactions (Mittler, 2002). It was worth noticing that BABA-induced accumulation of H2 O2 , an important defense signal, might be a synergistic consequence mediated by the above-mentioned antioxidant enzymes in mango fruit. CAT and APX isolated from tobacco have been identified as SA-binding proteins, and their enzymatic activities are inhibited by SA and its analogs (Chen et al., 1993; Durner and Klessing, 1995). Such binding and inhibition may incorporate SOD action to result in a relatively high level of H2 O2 accumulation in cells, thereby inducing resistance against pathogens via activation of defense genes and proteins in plants (Grant and Loake, 2000). Here, we speculate that the induced PAL activity in BABA-treated fruit might result in a higher accumulation of SA, which would subsequently interact with antioxidant enzymes and thus enhance the level of H2 O2 . In support of this idea, an exogenous SA-induced increase in H2 O2 production through mediating an enhancement of SOD and an inhibition of CAT and APX has been observed in sweet cherry and orange fruit (Chan and Tian, 2006; Huang et al., 2008). In conclusion, our results demonstrate that BABA treatment enhances resistance against anthracnose by inducing a defense response in infected mango fruit during storage. However, further study on the molecular mechanism of BABA-induced resistance by priming of defense reaction in mango fruit is needed. The study might also open new research directions aiming to explore the use of BABA as an elicitor in harvested commodities, although it has shown the differences in direct antifungal activity among microbes.

Acknowledgements This research was supported by the Fundamental Research Funds for Environment and Plant Protection Institute, CATAS (1630042012009; 2012hzs1J011) and Special Fund for Agroscientific Research in the Public Interest, China (201203092-2).

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