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Inhibition on anthracnose and induction of defense response by nitric oxide in pitaya fruit
Scientia Horticulturae 245 (2019) 224–230
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Inhibition on anthracnose and induction of defense response by nitric oxide in pitaya fruit
T
Meijiao Hua,b, Yingying Zhua,b, Gangshuai Liua, Zhaoyin Gaob, Min Lib, Zihan Sua, ⁎ Zhengke Zhanga, a b
College of Food Science and Technology, Hainan University, Haikou 570228, PR China Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pitaya Nitric oxide Induced resistance Fruit senescence Defense-related enzymes
The effect of nitric oxide (NO) on resistance of pitaya fruit against anthracnose caused by Colletotrichum gloeosporioides and its related mechanisms were investigated in this study. ‘Baiyulong’ pitaya fruit were immersed in 0.1 mM sodium nitroprusside (a NO donor) for 8 min, inoculated with spore suspension of C. gloeosporioides after 24 h of NO treatment, and then stored at 25 °C for up to 8 days. NO treatment markedly inhibited the lesion expansion on pathogen-inoculated pitaya fruit during storage. NO treatment also reduced the natural disease incidence and index of pitaya fruit stored at 25 °C. Furthermore, NO treatment increased the activities of defense-related enzymes including phenylalanine ammonia-lyase (PAL), CoA ligase (4CL), peroxidase (POD), polyphenol oxidase (PPO), chitinase (CHI) and β-1,3-glucanase (GLU), as well as elevated the contents of antifungal compounds including total phenolics, flavonoids and lignin. In addition, NO treatment reduced respiration rate and weight loss, while delayed the declines of firmness and soluble solids content (SSC). These results indicate that NO could effectively enhance the resistance of pitaya fruit to anthracnose, which might be ascribed to activation of defense responses and retardation of senescence.
1. Introduction Pitaya (Hylocereus undatus) is an important tropical and sub-tropical fruit due to its unique appearance, favorable flavor, rich health-promoting nutrients and high economic value (Fan et al., 2018). However, the pitaya fruit is highly vulnerable to infection by a variety of pathogens, resulting in deteriorated quality and restricted shelf-life (generally 5–7 d at ambient condition). Anthracnose caused by the fungus Colletotrichum gloeosporioides is considered as the most dominant postharvest disease of pitaya fruit (Ali et al., 2014). The C. gloeosporioides can either infect immature pitaya fruit in the field or attack the harvested fruit, with symptoms gradually occurring during postharvest storage (Ali et al., 2013). The postharvest disease of pitaya fruit can be well controlled by synthetic fungicides including benomyl, carbendazim, propineb and difenoconazole (Ali et al., 2013). Nevertheless, repeated and exclusive use of synthetic fungicides is liable to increase the resistance in pathogens, and cause the negative impacts on human health and environment. Thus, safe and eco-friendly approaches for controlling postharvest disease are required (Romanazzi et al., 2016). Induced resistance based on activating certain cellular defense
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responses by biotic or abiotic means is deemed a sustainable strategy for suppressing pathogens invasion and reducing postharvest diseases (Romanazzi et al., 2016). Nitric oxide (NO), a highly biological reactive free radical gas, functions as a signaling molecule that mediates numerous physiological and morphological processes in plants, ranging from growth, development, maturation, senescence to defense responses (Domingos et al., 2015). In postharvest application of NO, increasing evidence has indicated that exogenous NO treatment enhances resistance against pathogenic fungi in a variety of fruits. For example, NO treatments enhance resistance against brown rot caused by Monilinia fructicola in peaches (Li et al., 2017), against anthracnose caused by C. gloeosporioides in mangoes (Hu et al., 2014), and against gray mold caused by Botrytis cinerea in tomatoes (Zheng et al., 2011a). Enhanced resistance involves increases in activities of defense-related enzymes and their encoding genes expression, modulation of reactive oxygen species metabolism as well as delay of fruit ripening (Hu et al., 2014; Li et al., 2017; Zheng et al., 2017). In addition, Zheng et al. (2011b) observed that a fungal elicitor isolated from B. cinerea induced the disease resistance accompanying with a burst of endogenous NO and enhanced activities of NO synthase and nitric reductase (two key
Corresponding author. E-mail address: [email protected] (Z. Zhang).
https://doi.org/10.1016/j.scienta.2018.10.030 Received 28 November 2017; Received in revised form 27 August 2018; Accepted 14 October 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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percentage (%). Natural disease index was assessed using a rating scale based on the proportion of lesion area to the total surface area on each fruit. The scale was carried out as follows: 0 = no visible symptom; 1 = less than 5% decayed area; 2 = decayed area ranging from 5 to 15%; 3 = decayed area ranging from 16 to 25%; 4 = decayed area ranging from 26 to 50%, and 5 = more than 50% decayed area. The natural disease index was calculated using the formula: Σ (disease scale × number of fruit in each scale)/(number of total fruit × 5) × 100%. Each treatment contained three replicates, and 15 fruit per replicate.
enzymes activity related to NO generation in plants) in tomato fruit, and further treatments with NO quencher resulted in a block of resistance. These findings indicate that NO signaling plays a crucial role in systemic acquired resistance (SAR) and exogenous NO may confer protection against postharvest disease. However, there has been limited information available about the effects of postharvest NO treatment against C. gloeosporioides and its related defense mechanisms in pitaya fruit. The objective of current research was to study the influences of postharvest treatment with NO on anthracnose after inoculation with C. gloeosporioides in pitaya fruit during storage at 25 °C. Specific evaluations included the antifungal activity of NO against C. gloeosporioides in vivo, the activities of defense-related enzymes, the contents of antifungal compounds, and the effect on fruit senescence.
2.5. Sampling For assaying defense-related enzymes activity and antifungal compounds contents, the un-inoculated fruit peel tissue around the middle region was collected at 2-d interval, immediately frozen in liquid nitrogen, and then stored at −80 °C until use. There had three replicates for each treatment, with each replicate containing 6 fruit.
2. Materials and methods 2.1. Plant material and treatments Pitaya (H. undatus, cv. ‘Baiyulong’, a white-flesh cultivar) were obtained from an orchard in suburb of Danzhou city, Hainan province, China. Fruit at harvest were at a commercial maturity, with showing bright red-pink pericarp, plump bracts with green apex, bright white pulp with distinct seeds, and a good edible quality. Fruit were packed in corrugated fiberboard cartons (500 mm × 400 mm × 400 mm) with polyethylene film liner (0.03 mm thickness) and transported to laboratory at the same day using an air-conditioned cargo van at 25 °C and 55–60% relative humidity (RH). Fruit with uniformity of size, shape, color, and absence of disease and mechanical injury were selected.
2.6. Measurement of defense-related enzymes activity Frozen peel tissue (1 g) was homogenized with different extracting buffers (4 °C) using a pestle and mortal to obtain extracts for analyzing the following enzymes: 5 mL of 100 mM boric acid buffer (pH 8.8) containing 4% (w/v) polyvinylpyrrolidone (PVP), 5 mM β-mercaptoethanol and 2 mM ethylene diamine tetraacetic acid (EDTA) for phenylalanine ammonia lyase (PAL); 6 mL of 0.2 M Tris-HCl buffer (pH 8.0) containing 0.1 M dithiothreitol (DTT) and 25% (v/v) glecerol for 4coumarate: CoA ligase (4CL); 5 mL of 100 mM sodium acetate buffer (pH 7.0) containing 1 mM polyethyleneglycol (PEG), 4% poly-vinylpolypyrrolidone (PVPP) and 1% Triton-100 for peroxidase (POD) and polyphenol oxidase (PPO); 5 mL of 100 mM sodium acetate buffer (pH 5.2) containing 1 mM EDTA, 5 mM β-mercaptoethanol and 0.1% (w/v) ascorbic acid for chitinase (CHI) and β-1,3-glucanase (GLU). The extracts were centrifuged at 12,000g for 20 min at 4 °C. The supernatants were used for the analysis of enzymes activity. PAL activity was measured based on the increase in absorbance at 290 nm owing to the generation of trans-cinnamate from l-phenylalanine, following the method of Assis et al. (2001). One unit (U) of PAL activity was defined as the amount of enzyme causing an increase in absorbance of 0.01 per hour. 4-CL activity was measured by the increase in absorbance at 333 nm due to catalysis of substrates (CoA and p-coumaric acid) in the presence of enzyme and ATP into 4-coumarate: CoA, according to method of method of Yun et al. (2006). One unit (U) of 4-CL activity was defined as the amount of enzyme that caused an increase in absorbance of 0.01 per minute. POD activity was determined by the increase in absorbance at 470 nm owing to generation of tetraguaiacol from guaiacol under the presence of hydrogen peroxide, as described in method of Zhang et al. (2015). One unit (U) of enzyme activity was defined as the amount of enzyme that caused an increase in absorbance of 0.01 at 470 nm per minute. PPO activity was determined by the increase in absorbance at 420 nm using catechol solution as substrates (Chen et al., 2000). One unit (U) of PPO activity was defined as the amount of enzyme that caused an increase in absorbance of 0.001 per minute. CHI activity was measured following the method of Hu et al. (2014). One unit (U) of CHI activity was defined as the enzyme amount catalyzing the generation of 1 nmol N-acetyl-D-glucosamine from carboxymethylchitin per second. GLU activity was assayed using the method of Ippolito et al. (2000). One unit (U) of GLU activity was defined as the amount of enzyme catalyzing the laminarin into 1 nmol glucose equivalent per second. The activity of above-mentioned enzyme was expressed on U mg protein−1. Protein content was measured using the bicinchoninic acid
2.2. Pathogen C. gloeosporioides was isolated from infected pitaya fruit. After identification and purification, the pathogen was cultured on potato dextrose agar (PDA) at 28 °C for 7 d. Spore suspension (1 × 106 spores mL−1) was prepared using a hemocytometer according to our previous method (Zhang et al., 2013b). 2.3. Treatment and inoculation Pitaya fruit were assigned into 2 treatment groups, with 240 fruit per treatment group. The first group of fruit were immersed in 0.1 mM sodium nitroprusside (SNP, a NO donor) (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) solution for 8 min. The second group of fruit was treated with distilled water for 8 min, which was served as control. The optimal SNP concentration of 0.1 mM was employed based on a preliminary experiment using concentrations at 0.05, 0.1, 0.2 and 0.4 mM (data not shown). Twenty-four hours after treatment with SNP or water, 45 fruit of each treatment group were individually wound at the surface center of each fruit using a sterile cork borer (5 mm diameter), and thereafter a 20-μL of spore suspension of C. gloeosporioides was inoculated into the each wounded hole. All inoculated fruit for each treatment were subdivided into 3 lots (each lot represented a replicate, 15 fruit per replicate), and then were stored at ambient condition (25 ± 1 °C and 85–95% RH), and anthracnose severity as indicated by expansion of lesion diameter was measured every two days after inoculation. The remaining fruit without inoculation were stored at same condition to investigate natural disease incidence and physiobiochemical indexes at 2-d internal as described below. 2.4. Natural disease incidence and index Natural disease incidence was evaluated on 0–8 d of storage with 2d interval and calculated as the ratio of decayed fruit account for the total investigated fruit in each treatment, and was expressed as 225
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(BCA) method (Smith et al., 1985). 2.7. Determination of antifungal compounds content Peel tissue (1 g) was homogenized in 12 mL methanol (80%) using a pestle and mortar and then shaken in a water bath at 25 °C for 50 min. The homogenates were centrifuged at 12,000g for 15 min at 4 °C and the supernatant was used for measurement of total soluble phenolics and flavonoids. Total phenolics were assayed using the Folin-cioalteu procedure by Zhang et al. (2013a). Total phenolic content was calculated based on a standard curve of gallic acid, and was expressed as g kg−1 fresh weight (FW). Total flavonoids content was determined by an aluminum chloride colorimetric assay as reported in Jia et al. (1999). Total flavonoids content was calculated on basis of standard curve generated from catechin, which was expressed as g kg−1 FW. Lignin content was measured following the method of Morrison (1972), and the content was expressed as A 280 g−1 FW.
Fig. 1. Effect of NO treatment on lesion diameter of anthracnose in ‘Baiyulong’ pitaya fruit inoculated with C. gloeosporioides during storage at 25 °C. Vertical bars represent SE of the means of triplicate assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
2.8. Determination of firmness, respiration rate, SSC and weight loss Firmness of peeled pitaya fruit was determined using a FT-02 penetrometer (Effegi, Milan, Italy) with a 6 mm diameter probe, and firmness was expressed as Newtons (N). Respiration rate was measured using an infrared CO2 analyzer (model: GXH-3010D, Huayun Instrument Inc., Beijing, China), and expressed as CO2 production in mg kg−1 h−1. SSC in pitaya juice was measured using a Master-M refractometer (ATAGO Co. Ltd., Tokyo, Japan), and expressed as Brix. Weight loss was measured using a scale, and expressed as percentage (%) of the pitaya fruit weight at each time point relative to initial weight. For measurements of firmness, SSC and weigh loss, each treatment contained three replicates, with 6 fruit per replicate. For assay of respiration, there were 6 fruits in each treatment, and each fruit represented a biological replicate. 2.9. Statistical analysis Data were expressed as mean values ± standard error, and analyzed by one-way analysis of variance using SAS 8.1 software (SAS Institute Inc., Cary, NC, USA). Asterisks indicate significant difference between control and treatment at the same day (*P < 0.05). 3. Results 3.1. Anthracnose lesion diameter Anthracnose lesion in innoculated pitaya fruit did not expand within the initial 2 d of storage (Fig. 1). Afterwards, lesion diameters in pitaya fruit regardless of treatment gradually increased throughout storage (Fig. 1). NO treatment significantly suppressed the development of anthracnose, in which the lesion diameter of NO-treated fruit was averagely 21.6% lower than that in control fruit (Fig. 1).
Fig. 2. Effect of NO treatment on natural disease incidence (A) and index (B) in ‘Baiyulong’ pitaya fruit during storage at 25 °C. Vertical bars represent SE of the means of triplicate assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
3.2. Natural disease incidence and index Natural disease symptom in pitaya fruit began to occur after 4 d of storage (Fig. 2). NO treatment markedly reduced natural disease incidence and index during 4–8 d of storage when compared to control, with average value of both parameters from 4 to 8 d of storage in NOtreated fruit being 54.4% and 37.9% lower, respectively than that in control fruit (Fig. 2A and B).
then increased slowly to maximum at 6 d of storage, followed by a decrease in the rest of storage time (Fig. 3A). PAL activity in NO-treated fruit continuously increased to a maximum on 4 d, and thereafter exhibited a rapid decline (Fig. 3A). PAL activities in fruit receiving NO treatment were higher than those in control fruit throughout storage (Fig. 3A). 4-CL activity in control fruit sharply augmented to maximum at 6 d, and then decreased over rest of storage (Fig. 3B). 4-CL activity in fruit treated with NO showed a changing pattern similar to that in control
3.3. Defense-related enzymes activity PAL activity in control fruit slightly decreased in the initial 2 d and 226
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Fig. 3. Effect of NO treatment on activities of PAL (A), 4-CL (B), POD (C), PPO (D), CHI (E) and GLU (F) in ‘Baiyulong’ pitaya fruit during storage at 25 °C. Vertical bars represent SE of the means of triplicate assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
overall rise for total phenolic content occurred in NO-treated fruit (Fig. 4A). Comparing with control fruit, NO-treated fruit showed higher total phenolic contents at 2 and 6 d of storage (Fig. 4A). Control fruit exhibited a slight decrease in flavonoids content during storage (Fig. 4B). NO treatment resulted in a higher flavonoids accumulation compared with control fruit, except for 2 d of storage (Fig. 4B). Control fruit exhibited a slight increase in lignin content during storage (Fig. 4C). A much sharper increase in lignin content was noted in fruit receiving NO treatment, with values being higher than those in control fruit throughout storage (Fig. 4C).
fruit, but activities in NO-treated fruit were higher than those in control fruit during most of storage, except for on 4 d (Fig. 3B). POD activity in control pitaya fruit overall decreased throughout storage (Fig. 3C). NO-treated fruit exhibited a rise in POD activity within the initial 2 d, then a sharp decline during 2–4 d of storage, and thereafter a slight increase (Fig. 3C). NO-treated fruit exhibited higher POD activities compared with those in control fruit during most of storage, with the exception of 4 d of storage (Fig. 3C). Control fruit exhibited a slight decrease in PPO activity throughout storage (Fig. 3D). PPO activity in fruit treated with NO continuously increased within the initial 6 d of storage, and then maintained a steady level over the remaining storage time (Fig. 3D). The PPO activities in NO-treated fruit were higher than those in control fruit throughout storage (Fig. 3D). Both control and NO-treated fruit showed the changing trend of fluctuation for CHI activity (Fig. 3E). However, CHI activities in NOtreated fruit were higher than those in control fruit throughout storage (Fig. 3E). GLU activity in control fruit increased within the initial 2 d of storage, followed by maintaining a steady level over the rest of storage time (Fig. 3F). GLU activity in NO-treated fruit gradually rose, and reached to a maximum at 6 d, and then declined to a comparable level to that of control fruit at 8 d of storage (Fig. 3F).
3.5. Physiological parameters Firmness in control fruit sharply decreased throughout storage (Fig. 5A). NO-treated fruit exhibited a similar softening pattern to control fruit, but firmer than control fruit during the most of storage, except for on 8 d (Fig. 5A). Respiration rate in control fruit sharply declined within the first 4 d of storage, followed by an unchanged trend until the end of storage (Fig. 5B). NO treatment accelerated the decline in respiration rate, in which the rates were significantly lower than those in control fruit from 4 through 8 d (Fig. 5B). SSC in control fruit continuously decreased during storage (Fig. 5C). The trajectory of decline in SSC in NO-treated fruit was in parallel with that of control fruit, but remained higher levels in former throughout storage (Fig. 5C). Weight loss in both control and NO-treated fruit linearly increased throughout storage, but the slope rate in latter was lower than that of the former, except on 2 d (Fig. 5D).
3.4. Antifungal compounds contents Total phenolic content in control fruit rapidly decreased within the initial 2 d, followed by a slow increase during the remainder of storage (Fig. 4A). The total phenolic content at the end of storage (8 d) did not differ with initial level at 0 d (Fig. 4A). A trend of fluctuation but 227
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et al., 2014), citrus (Zhou et al., 2016); peaches (Gu et al., 2014; Li et al., 2017) and kiwifruits (Zheng et al., 2017). It has been well known that NO acts as a key gaseous messenger in plant-pathogen interactions (Bellin et al., 2013). The resistance afforded by NO in plants involves hypersensitive response and activation of salicylic acid (SA) mediated-signaling pathway (Domingos et al., 2015). SA signaling pathway generally relates to the activation of phenylpropanoid metabolism pathway and induction of pathogenesisrelated protein (PR) (Hu et al., 2014). PAL and 4-CL are key regulatory enzymes in the phenylpropanoid metabolism pathway, the former catalyzes L-phenylalanine to trans-cinnamic acid while the latter is responsible for formation of coumaric acid-CoA from various hydroxycinnamic acids (e.g. caffeic acid, coumaric acid and ferulic acid) (Singh et al., 2010). Increases in activity of phenylpropanoid metabolism enzymes account for accumulation of active antifungal metabolites such as phytoalexins, phenols, tannins, flavonoids and lignins, which may directly participate in defense responses through direct toxicity to pathogens, strengthening host cell structure and causing host hypersensitive reaction (Singh et al., 2010; Hu et al., 2014). POD is a key enzyme in the final stage of lignin biosynthesis, which catalyzes the polymerization of monomeric lignols into lignin that crosslink cell wall protein, contributing to consolidation of cell structure (Brisson et al., 1994; Zhang et al., 2011). PPO oxidizes phenols into fungitoxic quinines that may directly inhibit pathogens growth (Liu et al., 2014; Ge et al., 2017). CHI and GLU are two PR proteins that play crucial roles in defense against pathogens infection in plants. CHI functions in decomposing chitin that constitutes the main component of fungal cell wall (Wang et al., 2011). GLU executes its antifungal role by hydrolyzing β-1,3linked glucans, which are the second major constituents of the fungal cell wall (Wang et al., 2011). Also, released oligosaccharides by GLU may be served as non-specific biological elicitors to induce defense reaction (Zhang et al., 2011). The present results demonstrated that NO treatment enhanced the activities of defense-related enzymes including PAL, 4CL, POD, CHI and GLU, and promoted the accumulation of antifungal compounds including total phenolics, flavonoids and lignin, which might contribute to the suppression on postharvest anthracnose and natural disease in pitaya fruit during storage. Similarly, Li et al. (2017) reported that NO inhibited brown rot caused by M. fructicola in harvested peach fruit, which was correlated with increases in the genes expression and activities of phenylpropanoid metabolism enzymes including PAL, cinnamate-4-hydroxylase (C4H), 4CL, chalcone synthase (CHS) and chalcone isomerase (CHI), in paralell with accumulation of the total phenolics, flavonoids and anthocyanins. Gu et al. (2014) noted that NO treatment enhanced the expression of several PR protein genes including CHI, GLU, PR-1 and PR-10, accounting for the increased resistance against brown rot in harvested peaches, and contrarily, such induction of PR protein genes and acquired resistance due to NO were abolished by an application of NO scavenger (cPTIO), indicating an important role of endogenous NO as a signaling molecule in SAR. In addition, enhanced resistance against postharvest anthracnose in relation to higher PAL, POD and PPO activities were also observed in chitosan-treated pitaya fruit (Zahid et al., 2015). Pitaya is a non-climacteric fruit generally characterized by decreased respiration rate, fruit firmness and soluble solids and increased weight loss during fruit senescence (Ali et al., 2013). The data presented here exhibited that NO treatment markedly reduced the respiration rate and weight loss, and delayed the decrease in firmness and SSC in ‘Baiyulong’ pitaya fruit during storage at 25 °C. These results suggest that NO might delay the development of senescence and maintain the quality characteristics of pitaya fruit. In line with our findings, NO treatment was also observed to exert positive effects against ripening and/or senescence in a variety of harvested fruits including strawberry (Wills et al., 2000), jujube (Zhu et al., 2009), mango (Hu et al., 2014) and litchi (Barman et al., 2014). It has been well known that delay of ripening and senescence in response to NO in
Fig. 4. Effect of NO treatment on contents of total phenolics (A), flavonoids (B) and lignin (C), in ‘Baiyulong’ pitaya fruit during storage at 25 °C. Vertical bars represent SE of the means of triplicate assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
4. Discussion Postharvest decay caused by anthracnose is a main limit on maintaining the quality of pitaya fruit during shelf life (Awang et al., 2011). To reduce occurrence of anthracnose disease in pitaya fruit, a few postharvest approaches as alternatives to fungicides, such as hot air treatment (Hoa et al., 2006), calcium chloride treatment (Awang et al., 2011) and submicron chitosan coating (Ali et al., 2014; Zahid et al., 2015) have been tested. In our study, we observed that postharvest treatment with NO by means of immersion in 0.1 mM aqueous SNP significantly inhibited the expansion of anthracnose lesion in ‘Baiyulong’ pitaya fruit inoculated with C. gloeosporiodides, and reduced the natural disease incidence and severity during storage at ambient condition. Provided protection against pathogens infection due to NO treatment was also reported in other postharvest fruits such as tomatoes (Fan et al., 2008; Lai et al., 2011; Zheng et al., 2011a), mangoes (Hu 228
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Fig. 5. Effect of NO treatment on firmness (A), respiration rate (B), SSC (C) and weight loss (D) in ‘Baiyulong’ pitaya fruit during storage at 25 °C. Vertical bars represent SE of the means of triplicate (for firmness, SSC and weight loss) or sextuplicate (for respiration rate) assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
climacteric fruit involves inhibition of ethylene mode of action (Singh et al., 2013). In non-climacteric fruit, for example in litchi, it was noted that NO inhibited browning and maintained the quality attributes, which might be attributed to enhanced antioxidant capacity (Barman et al., 2014). In this study, NO treatment stimulated accumulations of total phenolics and flavonoids that are classed as antifungal metabolites as well as antioxidants, which seemed plausible to contribute to the delay of senescence in pitaya fruit. However, the explicit mode of action through which exposure to NO might inhibit senescence-related processes in non-climacteric fruit is not yet well clarified, which warrants further study. In general, significant physio-biochemical changes during fruit ripening and senescence may lead to reduced resistance to pathogens (Hu et al., 2014). Accordingly, we propose that reduction of postharvest anthracnose in pitaya fruit by NO might be not only due to activation of defense mechanisms but also ascribed to general delay of senescence. In summary, the present results demonstrated that NO treatment effectively enhanced resistance of pitaya fruit to postharvest anthracnose caused by C. gloeosporioides. The enhanced resistance might be associated with retardation of senescence as well as activation of defense mechanisms including increases in defense-related enzymes activity and antifungal compounds content. We propose that NO treatment could be a potential approach for controlling disease and maintaining quality of harvested pitaya fruit.
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Acknowledgements The study was supported by the National Natural Science Foundation of China (31300575, 31560472), and the Scientific Research Funds of Hainan University, China (kyqd-1550). References Ali, A., Zahid, N., Manickam, N., Siddiqui, Y., Alderson, P.G., Maqbool, M., 2013. Effectiveness of submicron chitosan dispersions in controlling anthracnose and maintaining quality of dragon fruit. Postharvest Biol. Technol. 86, 147–153. Ali, A., Zahid, N., Manickam, S., Siddiqui, Y., Alderson, P.G., Maqbool, M., 2014. Induction of lignin and pathogenesis related proteins in dragon fruit plants in response to submicron chitosan dispersions. Crop Prot. 63, 83–88. Assis, J.S., Maldonado, R., Munoz, T., Escribano, M.I., Merodio, C., 2001. Effect of highcarbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biol. Technol. 23, 33–39.
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Inhibition on anthracnose and induction of defense response by nitric oxide in pitaya fruit
T
Meijiao Hua,b, Yingying Zhua,b, Gangshuai Liua, Zhaoyin Gaob, Min Lib, Zihan Sua, ⁎ Zhengke Zhanga, a b
College of Food Science and Technology, Hainan University, Haikou 570228, PR China Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pitaya Nitric oxide Induced resistance Fruit senescence Defense-related enzymes
The effect of nitric oxide (NO) on resistance of pitaya fruit against anthracnose caused by Colletotrichum gloeosporioides and its related mechanisms were investigated in this study. ‘Baiyulong’ pitaya fruit were immersed in 0.1 mM sodium nitroprusside (a NO donor) for 8 min, inoculated with spore suspension of C. gloeosporioides after 24 h of NO treatment, and then stored at 25 °C for up to 8 days. NO treatment markedly inhibited the lesion expansion on pathogen-inoculated pitaya fruit during storage. NO treatment also reduced the natural disease incidence and index of pitaya fruit stored at 25 °C. Furthermore, NO treatment increased the activities of defense-related enzymes including phenylalanine ammonia-lyase (PAL), CoA ligase (4CL), peroxidase (POD), polyphenol oxidase (PPO), chitinase (CHI) and β-1,3-glucanase (GLU), as well as elevated the contents of antifungal compounds including total phenolics, flavonoids and lignin. In addition, NO treatment reduced respiration rate and weight loss, while delayed the declines of firmness and soluble solids content (SSC). These results indicate that NO could effectively enhance the resistance of pitaya fruit to anthracnose, which might be ascribed to activation of defense responses and retardation of senescence.
1. Introduction Pitaya (Hylocereus undatus) is an important tropical and sub-tropical fruit due to its unique appearance, favorable flavor, rich health-promoting nutrients and high economic value (Fan et al., 2018). However, the pitaya fruit is highly vulnerable to infection by a variety of pathogens, resulting in deteriorated quality and restricted shelf-life (generally 5–7 d at ambient condition). Anthracnose caused by the fungus Colletotrichum gloeosporioides is considered as the most dominant postharvest disease of pitaya fruit (Ali et al., 2014). The C. gloeosporioides can either infect immature pitaya fruit in the field or attack the harvested fruit, with symptoms gradually occurring during postharvest storage (Ali et al., 2013). The postharvest disease of pitaya fruit can be well controlled by synthetic fungicides including benomyl, carbendazim, propineb and difenoconazole (Ali et al., 2013). Nevertheless, repeated and exclusive use of synthetic fungicides is liable to increase the resistance in pathogens, and cause the negative impacts on human health and environment. Thus, safe and eco-friendly approaches for controlling postharvest disease are required (Romanazzi et al., 2016). Induced resistance based on activating certain cellular defense
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responses by biotic or abiotic means is deemed a sustainable strategy for suppressing pathogens invasion and reducing postharvest diseases (Romanazzi et al., 2016). Nitric oxide (NO), a highly biological reactive free radical gas, functions as a signaling molecule that mediates numerous physiological and morphological processes in plants, ranging from growth, development, maturation, senescence to defense responses (Domingos et al., 2015). In postharvest application of NO, increasing evidence has indicated that exogenous NO treatment enhances resistance against pathogenic fungi in a variety of fruits. For example, NO treatments enhance resistance against brown rot caused by Monilinia fructicola in peaches (Li et al., 2017), against anthracnose caused by C. gloeosporioides in mangoes (Hu et al., 2014), and against gray mold caused by Botrytis cinerea in tomatoes (Zheng et al., 2011a). Enhanced resistance involves increases in activities of defense-related enzymes and their encoding genes expression, modulation of reactive oxygen species metabolism as well as delay of fruit ripening (Hu et al., 2014; Li et al., 2017; Zheng et al., 2017). In addition, Zheng et al. (2011b) observed that a fungal elicitor isolated from B. cinerea induced the disease resistance accompanying with a burst of endogenous NO and enhanced activities of NO synthase and nitric reductase (two key
Corresponding author. E-mail address: [email protected] (Z. Zhang).
https://doi.org/10.1016/j.scienta.2018.10.030 Received 28 November 2017; Received in revised form 27 August 2018; Accepted 14 October 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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percentage (%). Natural disease index was assessed using a rating scale based on the proportion of lesion area to the total surface area on each fruit. The scale was carried out as follows: 0 = no visible symptom; 1 = less than 5% decayed area; 2 = decayed area ranging from 5 to 15%; 3 = decayed area ranging from 16 to 25%; 4 = decayed area ranging from 26 to 50%, and 5 = more than 50% decayed area. The natural disease index was calculated using the formula: Σ (disease scale × number of fruit in each scale)/(number of total fruit × 5) × 100%. Each treatment contained three replicates, and 15 fruit per replicate.
enzymes activity related to NO generation in plants) in tomato fruit, and further treatments with NO quencher resulted in a block of resistance. These findings indicate that NO signaling plays a crucial role in systemic acquired resistance (SAR) and exogenous NO may confer protection against postharvest disease. However, there has been limited information available about the effects of postharvest NO treatment against C. gloeosporioides and its related defense mechanisms in pitaya fruit. The objective of current research was to study the influences of postharvest treatment with NO on anthracnose after inoculation with C. gloeosporioides in pitaya fruit during storage at 25 °C. Specific evaluations included the antifungal activity of NO against C. gloeosporioides in vivo, the activities of defense-related enzymes, the contents of antifungal compounds, and the effect on fruit senescence.
2.5. Sampling For assaying defense-related enzymes activity and antifungal compounds contents, the un-inoculated fruit peel tissue around the middle region was collected at 2-d interval, immediately frozen in liquid nitrogen, and then stored at −80 °C until use. There had three replicates for each treatment, with each replicate containing 6 fruit.
2. Materials and methods 2.1. Plant material and treatments Pitaya (H. undatus, cv. ‘Baiyulong’, a white-flesh cultivar) were obtained from an orchard in suburb of Danzhou city, Hainan province, China. Fruit at harvest were at a commercial maturity, with showing bright red-pink pericarp, plump bracts with green apex, bright white pulp with distinct seeds, and a good edible quality. Fruit were packed in corrugated fiberboard cartons (500 mm × 400 mm × 400 mm) with polyethylene film liner (0.03 mm thickness) and transported to laboratory at the same day using an air-conditioned cargo van at 25 °C and 55–60% relative humidity (RH). Fruit with uniformity of size, shape, color, and absence of disease and mechanical injury were selected.
2.6. Measurement of defense-related enzymes activity Frozen peel tissue (1 g) was homogenized with different extracting buffers (4 °C) using a pestle and mortal to obtain extracts for analyzing the following enzymes: 5 mL of 100 mM boric acid buffer (pH 8.8) containing 4% (w/v) polyvinylpyrrolidone (PVP), 5 mM β-mercaptoethanol and 2 mM ethylene diamine tetraacetic acid (EDTA) for phenylalanine ammonia lyase (PAL); 6 mL of 0.2 M Tris-HCl buffer (pH 8.0) containing 0.1 M dithiothreitol (DTT) and 25% (v/v) glecerol for 4coumarate: CoA ligase (4CL); 5 mL of 100 mM sodium acetate buffer (pH 7.0) containing 1 mM polyethyleneglycol (PEG), 4% poly-vinylpolypyrrolidone (PVPP) and 1% Triton-100 for peroxidase (POD) and polyphenol oxidase (PPO); 5 mL of 100 mM sodium acetate buffer (pH 5.2) containing 1 mM EDTA, 5 mM β-mercaptoethanol and 0.1% (w/v) ascorbic acid for chitinase (CHI) and β-1,3-glucanase (GLU). The extracts were centrifuged at 12,000g for 20 min at 4 °C. The supernatants were used for the analysis of enzymes activity. PAL activity was measured based on the increase in absorbance at 290 nm owing to the generation of trans-cinnamate from l-phenylalanine, following the method of Assis et al. (2001). One unit (U) of PAL activity was defined as the amount of enzyme causing an increase in absorbance of 0.01 per hour. 4-CL activity was measured by the increase in absorbance at 333 nm due to catalysis of substrates (CoA and p-coumaric acid) in the presence of enzyme and ATP into 4-coumarate: CoA, according to method of method of Yun et al. (2006). One unit (U) of 4-CL activity was defined as the amount of enzyme that caused an increase in absorbance of 0.01 per minute. POD activity was determined by the increase in absorbance at 470 nm owing to generation of tetraguaiacol from guaiacol under the presence of hydrogen peroxide, as described in method of Zhang et al. (2015). One unit (U) of enzyme activity was defined as the amount of enzyme that caused an increase in absorbance of 0.01 at 470 nm per minute. PPO activity was determined by the increase in absorbance at 420 nm using catechol solution as substrates (Chen et al., 2000). One unit (U) of PPO activity was defined as the amount of enzyme that caused an increase in absorbance of 0.001 per minute. CHI activity was measured following the method of Hu et al. (2014). One unit (U) of CHI activity was defined as the enzyme amount catalyzing the generation of 1 nmol N-acetyl-D-glucosamine from carboxymethylchitin per second. GLU activity was assayed using the method of Ippolito et al. (2000). One unit (U) of GLU activity was defined as the amount of enzyme catalyzing the laminarin into 1 nmol glucose equivalent per second. The activity of above-mentioned enzyme was expressed on U mg protein−1. Protein content was measured using the bicinchoninic acid
2.2. Pathogen C. gloeosporioides was isolated from infected pitaya fruit. After identification and purification, the pathogen was cultured on potato dextrose agar (PDA) at 28 °C for 7 d. Spore suspension (1 × 106 spores mL−1) was prepared using a hemocytometer according to our previous method (Zhang et al., 2013b). 2.3. Treatment and inoculation Pitaya fruit were assigned into 2 treatment groups, with 240 fruit per treatment group. The first group of fruit were immersed in 0.1 mM sodium nitroprusside (SNP, a NO donor) (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) solution for 8 min. The second group of fruit was treated with distilled water for 8 min, which was served as control. The optimal SNP concentration of 0.1 mM was employed based on a preliminary experiment using concentrations at 0.05, 0.1, 0.2 and 0.4 mM (data not shown). Twenty-four hours after treatment with SNP or water, 45 fruit of each treatment group were individually wound at the surface center of each fruit using a sterile cork borer (5 mm diameter), and thereafter a 20-μL of spore suspension of C. gloeosporioides was inoculated into the each wounded hole. All inoculated fruit for each treatment were subdivided into 3 lots (each lot represented a replicate, 15 fruit per replicate), and then were stored at ambient condition (25 ± 1 °C and 85–95% RH), and anthracnose severity as indicated by expansion of lesion diameter was measured every two days after inoculation. The remaining fruit without inoculation were stored at same condition to investigate natural disease incidence and physiobiochemical indexes at 2-d internal as described below. 2.4. Natural disease incidence and index Natural disease incidence was evaluated on 0–8 d of storage with 2d interval and calculated as the ratio of decayed fruit account for the total investigated fruit in each treatment, and was expressed as 225
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(BCA) method (Smith et al., 1985). 2.7. Determination of antifungal compounds content Peel tissue (1 g) was homogenized in 12 mL methanol (80%) using a pestle and mortar and then shaken in a water bath at 25 °C for 50 min. The homogenates were centrifuged at 12,000g for 15 min at 4 °C and the supernatant was used for measurement of total soluble phenolics and flavonoids. Total phenolics were assayed using the Folin-cioalteu procedure by Zhang et al. (2013a). Total phenolic content was calculated based on a standard curve of gallic acid, and was expressed as g kg−1 fresh weight (FW). Total flavonoids content was determined by an aluminum chloride colorimetric assay as reported in Jia et al. (1999). Total flavonoids content was calculated on basis of standard curve generated from catechin, which was expressed as g kg−1 FW. Lignin content was measured following the method of Morrison (1972), and the content was expressed as A 280 g−1 FW.
Fig. 1. Effect of NO treatment on lesion diameter of anthracnose in ‘Baiyulong’ pitaya fruit inoculated with C. gloeosporioides during storage at 25 °C. Vertical bars represent SE of the means of triplicate assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
2.8. Determination of firmness, respiration rate, SSC and weight loss Firmness of peeled pitaya fruit was determined using a FT-02 penetrometer (Effegi, Milan, Italy) with a 6 mm diameter probe, and firmness was expressed as Newtons (N). Respiration rate was measured using an infrared CO2 analyzer (model: GXH-3010D, Huayun Instrument Inc., Beijing, China), and expressed as CO2 production in mg kg−1 h−1. SSC in pitaya juice was measured using a Master-M refractometer (ATAGO Co. Ltd., Tokyo, Japan), and expressed as Brix. Weight loss was measured using a scale, and expressed as percentage (%) of the pitaya fruit weight at each time point relative to initial weight. For measurements of firmness, SSC and weigh loss, each treatment contained three replicates, with 6 fruit per replicate. For assay of respiration, there were 6 fruits in each treatment, and each fruit represented a biological replicate. 2.9. Statistical analysis Data were expressed as mean values ± standard error, and analyzed by one-way analysis of variance using SAS 8.1 software (SAS Institute Inc., Cary, NC, USA). Asterisks indicate significant difference between control and treatment at the same day (*P < 0.05). 3. Results 3.1. Anthracnose lesion diameter Anthracnose lesion in innoculated pitaya fruit did not expand within the initial 2 d of storage (Fig. 1). Afterwards, lesion diameters in pitaya fruit regardless of treatment gradually increased throughout storage (Fig. 1). NO treatment significantly suppressed the development of anthracnose, in which the lesion diameter of NO-treated fruit was averagely 21.6% lower than that in control fruit (Fig. 1).
Fig. 2. Effect of NO treatment on natural disease incidence (A) and index (B) in ‘Baiyulong’ pitaya fruit during storage at 25 °C. Vertical bars represent SE of the means of triplicate assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
3.2. Natural disease incidence and index Natural disease symptom in pitaya fruit began to occur after 4 d of storage (Fig. 2). NO treatment markedly reduced natural disease incidence and index during 4–8 d of storage when compared to control, with average value of both parameters from 4 to 8 d of storage in NOtreated fruit being 54.4% and 37.9% lower, respectively than that in control fruit (Fig. 2A and B).
then increased slowly to maximum at 6 d of storage, followed by a decrease in the rest of storage time (Fig. 3A). PAL activity in NO-treated fruit continuously increased to a maximum on 4 d, and thereafter exhibited a rapid decline (Fig. 3A). PAL activities in fruit receiving NO treatment were higher than those in control fruit throughout storage (Fig. 3A). 4-CL activity in control fruit sharply augmented to maximum at 6 d, and then decreased over rest of storage (Fig. 3B). 4-CL activity in fruit treated with NO showed a changing pattern similar to that in control
3.3. Defense-related enzymes activity PAL activity in control fruit slightly decreased in the initial 2 d and 226
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Fig. 3. Effect of NO treatment on activities of PAL (A), 4-CL (B), POD (C), PPO (D), CHI (E) and GLU (F) in ‘Baiyulong’ pitaya fruit during storage at 25 °C. Vertical bars represent SE of the means of triplicate assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
overall rise for total phenolic content occurred in NO-treated fruit (Fig. 4A). Comparing with control fruit, NO-treated fruit showed higher total phenolic contents at 2 and 6 d of storage (Fig. 4A). Control fruit exhibited a slight decrease in flavonoids content during storage (Fig. 4B). NO treatment resulted in a higher flavonoids accumulation compared with control fruit, except for 2 d of storage (Fig. 4B). Control fruit exhibited a slight increase in lignin content during storage (Fig. 4C). A much sharper increase in lignin content was noted in fruit receiving NO treatment, with values being higher than those in control fruit throughout storage (Fig. 4C).
fruit, but activities in NO-treated fruit were higher than those in control fruit during most of storage, except for on 4 d (Fig. 3B). POD activity in control pitaya fruit overall decreased throughout storage (Fig. 3C). NO-treated fruit exhibited a rise in POD activity within the initial 2 d, then a sharp decline during 2–4 d of storage, and thereafter a slight increase (Fig. 3C). NO-treated fruit exhibited higher POD activities compared with those in control fruit during most of storage, with the exception of 4 d of storage (Fig. 3C). Control fruit exhibited a slight decrease in PPO activity throughout storage (Fig. 3D). PPO activity in fruit treated with NO continuously increased within the initial 6 d of storage, and then maintained a steady level over the remaining storage time (Fig. 3D). The PPO activities in NO-treated fruit were higher than those in control fruit throughout storage (Fig. 3D). Both control and NO-treated fruit showed the changing trend of fluctuation for CHI activity (Fig. 3E). However, CHI activities in NOtreated fruit were higher than those in control fruit throughout storage (Fig. 3E). GLU activity in control fruit increased within the initial 2 d of storage, followed by maintaining a steady level over the rest of storage time (Fig. 3F). GLU activity in NO-treated fruit gradually rose, and reached to a maximum at 6 d, and then declined to a comparable level to that of control fruit at 8 d of storage (Fig. 3F).
3.5. Physiological parameters Firmness in control fruit sharply decreased throughout storage (Fig. 5A). NO-treated fruit exhibited a similar softening pattern to control fruit, but firmer than control fruit during the most of storage, except for on 8 d (Fig. 5A). Respiration rate in control fruit sharply declined within the first 4 d of storage, followed by an unchanged trend until the end of storage (Fig. 5B). NO treatment accelerated the decline in respiration rate, in which the rates were significantly lower than those in control fruit from 4 through 8 d (Fig. 5B). SSC in control fruit continuously decreased during storage (Fig. 5C). The trajectory of decline in SSC in NO-treated fruit was in parallel with that of control fruit, but remained higher levels in former throughout storage (Fig. 5C). Weight loss in both control and NO-treated fruit linearly increased throughout storage, but the slope rate in latter was lower than that of the former, except on 2 d (Fig. 5D).
3.4. Antifungal compounds contents Total phenolic content in control fruit rapidly decreased within the initial 2 d, followed by a slow increase during the remainder of storage (Fig. 4A). The total phenolic content at the end of storage (8 d) did not differ with initial level at 0 d (Fig. 4A). A trend of fluctuation but 227
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et al., 2014), citrus (Zhou et al., 2016); peaches (Gu et al., 2014; Li et al., 2017) and kiwifruits (Zheng et al., 2017). It has been well known that NO acts as a key gaseous messenger in plant-pathogen interactions (Bellin et al., 2013). The resistance afforded by NO in plants involves hypersensitive response and activation of salicylic acid (SA) mediated-signaling pathway (Domingos et al., 2015). SA signaling pathway generally relates to the activation of phenylpropanoid metabolism pathway and induction of pathogenesisrelated protein (PR) (Hu et al., 2014). PAL and 4-CL are key regulatory enzymes in the phenylpropanoid metabolism pathway, the former catalyzes L-phenylalanine to trans-cinnamic acid while the latter is responsible for formation of coumaric acid-CoA from various hydroxycinnamic acids (e.g. caffeic acid, coumaric acid and ferulic acid) (Singh et al., 2010). Increases in activity of phenylpropanoid metabolism enzymes account for accumulation of active antifungal metabolites such as phytoalexins, phenols, tannins, flavonoids and lignins, which may directly participate in defense responses through direct toxicity to pathogens, strengthening host cell structure and causing host hypersensitive reaction (Singh et al., 2010; Hu et al., 2014). POD is a key enzyme in the final stage of lignin biosynthesis, which catalyzes the polymerization of monomeric lignols into lignin that crosslink cell wall protein, contributing to consolidation of cell structure (Brisson et al., 1994; Zhang et al., 2011). PPO oxidizes phenols into fungitoxic quinines that may directly inhibit pathogens growth (Liu et al., 2014; Ge et al., 2017). CHI and GLU are two PR proteins that play crucial roles in defense against pathogens infection in plants. CHI functions in decomposing chitin that constitutes the main component of fungal cell wall (Wang et al., 2011). GLU executes its antifungal role by hydrolyzing β-1,3linked glucans, which are the second major constituents of the fungal cell wall (Wang et al., 2011). Also, released oligosaccharides by GLU may be served as non-specific biological elicitors to induce defense reaction (Zhang et al., 2011). The present results demonstrated that NO treatment enhanced the activities of defense-related enzymes including PAL, 4CL, POD, CHI and GLU, and promoted the accumulation of antifungal compounds including total phenolics, flavonoids and lignin, which might contribute to the suppression on postharvest anthracnose and natural disease in pitaya fruit during storage. Similarly, Li et al. (2017) reported that NO inhibited brown rot caused by M. fructicola in harvested peach fruit, which was correlated with increases in the genes expression and activities of phenylpropanoid metabolism enzymes including PAL, cinnamate-4-hydroxylase (C4H), 4CL, chalcone synthase (CHS) and chalcone isomerase (CHI), in paralell with accumulation of the total phenolics, flavonoids and anthocyanins. Gu et al. (2014) noted that NO treatment enhanced the expression of several PR protein genes including CHI, GLU, PR-1 and PR-10, accounting for the increased resistance against brown rot in harvested peaches, and contrarily, such induction of PR protein genes and acquired resistance due to NO were abolished by an application of NO scavenger (cPTIO), indicating an important role of endogenous NO as a signaling molecule in SAR. In addition, enhanced resistance against postharvest anthracnose in relation to higher PAL, POD and PPO activities were also observed in chitosan-treated pitaya fruit (Zahid et al., 2015). Pitaya is a non-climacteric fruit generally characterized by decreased respiration rate, fruit firmness and soluble solids and increased weight loss during fruit senescence (Ali et al., 2013). The data presented here exhibited that NO treatment markedly reduced the respiration rate and weight loss, and delayed the decrease in firmness and SSC in ‘Baiyulong’ pitaya fruit during storage at 25 °C. These results suggest that NO might delay the development of senescence and maintain the quality characteristics of pitaya fruit. In line with our findings, NO treatment was also observed to exert positive effects against ripening and/or senescence in a variety of harvested fruits including strawberry (Wills et al., 2000), jujube (Zhu et al., 2009), mango (Hu et al., 2014) and litchi (Barman et al., 2014). It has been well known that delay of ripening and senescence in response to NO in
Fig. 4. Effect of NO treatment on contents of total phenolics (A), flavonoids (B) and lignin (C), in ‘Baiyulong’ pitaya fruit during storage at 25 °C. Vertical bars represent SE of the means of triplicate assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
4. Discussion Postharvest decay caused by anthracnose is a main limit on maintaining the quality of pitaya fruit during shelf life (Awang et al., 2011). To reduce occurrence of anthracnose disease in pitaya fruit, a few postharvest approaches as alternatives to fungicides, such as hot air treatment (Hoa et al., 2006), calcium chloride treatment (Awang et al., 2011) and submicron chitosan coating (Ali et al., 2014; Zahid et al., 2015) have been tested. In our study, we observed that postharvest treatment with NO by means of immersion in 0.1 mM aqueous SNP significantly inhibited the expansion of anthracnose lesion in ‘Baiyulong’ pitaya fruit inoculated with C. gloeosporiodides, and reduced the natural disease incidence and severity during storage at ambient condition. Provided protection against pathogens infection due to NO treatment was also reported in other postharvest fruits such as tomatoes (Fan et al., 2008; Lai et al., 2011; Zheng et al., 2011a), mangoes (Hu 228
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Fig. 5. Effect of NO treatment on firmness (A), respiration rate (B), SSC (C) and weight loss (D) in ‘Baiyulong’ pitaya fruit during storage at 25 °C. Vertical bars represent SE of the means of triplicate (for firmness, SSC and weight loss) or sextuplicate (for respiration rate) assays. Asterisks represent that values are significantly different between control and NO-treated fruit at the same day (* P < 0.05).
climacteric fruit involves inhibition of ethylene mode of action (Singh et al., 2013). In non-climacteric fruit, for example in litchi, it was noted that NO inhibited browning and maintained the quality attributes, which might be attributed to enhanced antioxidant capacity (Barman et al., 2014). In this study, NO treatment stimulated accumulations of total phenolics and flavonoids that are classed as antifungal metabolites as well as antioxidants, which seemed plausible to contribute to the delay of senescence in pitaya fruit. However, the explicit mode of action through which exposure to NO might inhibit senescence-related processes in non-climacteric fruit is not yet well clarified, which warrants further study. In general, significant physio-biochemical changes during fruit ripening and senescence may lead to reduced resistance to pathogens (Hu et al., 2014). Accordingly, we propose that reduction of postharvest anthracnose in pitaya fruit by NO might be not only due to activation of defense mechanisms but also ascribed to general delay of senescence. In summary, the present results demonstrated that NO treatment effectively enhanced resistance of pitaya fruit to postharvest anthracnose caused by C. gloeosporioides. The enhanced resistance might be associated with retardation of senescence as well as activation of defense mechanisms including increases in defense-related enzymes activity and antifungal compounds content. We propose that NO treatment could be a potential approach for controlling disease and maintaining quality of harvested pitaya fruit.
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