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Antifungal efficacy of ursolic acid in control of Alternaria alternata causing black spot rot on apple fruit and possible mechanisms involved
Scientia Horticulturae 256 (2019) 108636
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Antifungal efficacy of ursolic acid in control of Alternaria alternata causing black spot rot on apple fruit and possible mechanisms involved Chang Shu, Handong Zhao, Wenxiao Jiao, Bangdi Liu, Jiankang Cao, Weibo Jiang
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College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Qinghuadonglu Road, Beijing 100083, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Ursolic acid Black spot rot Apple fruit Alternaria alternata Antifungal activity
Black spot rot caused by Alternaria alternata is becoming a main postharvest disease on apple fruit. The objective of this study was to elucidate the antifungal efficiency of ursolic acid (UA) against A. alternata and its application in preserving apple fruit. The results showed that UA induced disturbance of membrane permeability and integrity, as well as intracellular ROS accumulation in A. alternata, result in the sabotaged morphology and lysis of the pathogen, thus exert directly antifungal activity. In addition, the application of UA significantly inhibited the black spot rot development on apple fruit, the increased defense enzyme activities were detected. These results suggested that, in addition to its direct antifungal activity, UA induced the defense responses in apple fruit against the postharvest pathogen invasion. UA is widely distributed in the plant kingdom and could be a promising alternative for Alternata diseases management.
1. Introduction Apple (Malus domestica Borkh.) fruit is one of the most cultivated and consumed fruit crops worldwide. The recent statistics from FAO (2017) showed that China is the largest apple producer. Apple fruits are mostly preserved in cold condition for long-term preserving and marketing (Shen et al., 2018). However, despite modern storage facilities, postharvest losses are still ubiquitous. Fungal pathogens are mainly responsible for significant economic losses. Black spot rot caused by Alternaria alternata is becoming an important postharvest disease on apple fruit in China since the widespread implementation of the practice of bagging apples during the period of fruit development (Dang et al., 2018; Ge et al., 2019a). This may be attributed to the higher temperature and humidity inside the bag, which made pathogens more possible to grow. A. alternata is a latent fungus that could contaminate the fruits through breaks in the skin or other weakened areas in the orchard and during the storage (Jijakli and Lepoivre, 2006) and could rapidly develop at low temperatures and cause tremendous postharvest losses during storage period and shelf life (Yan et al., 2015). Meanwhile, A. alternata can yield secondary metabolites considered as both phytotoxins and mycotoxins, which can be detrimental to humans, causing food safety issues (Wang et al., 2017). The persistent application of synthetic fungicides such as iprodione, tebuconazole, and mancozeb has gradually induced the emergence of fungicide-resistant strains as well as the environmental contamination, while the public
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has begun to concern more about the harmful effects of synthetic fungicides on human health and related negative effects (Yuan et al., 2019). Therefore, there is an urgent need to develop an effective and environmentally friendly alternative to these traditional fungicides to control black spot rot on apple fruit. The use of natural botanical ingredients is thought to be a good viable alternative to control postharvest diseases. Plant metabolites have long been used as preservatives in food preservation and traditional medicine (da Rocha Neto et al., 2019d), it has been reported that essential oils (Castro et al., 2017; Xu et al., 2018), phenolic acids and its derivatives (Li et al., 2018a,b; Shaik et al., 2016) as well as terpenoids (Jabeen et al., 2011; Mahlo et al., 2013; Mahlo and Eloff, 2014) could inhibit the growth of A. alternata and other postharvest pathogens and would be applied as natural fungicide for control postharvest decay. Among the compounds studied above, triterpenoids have attracted the attention among researchers. Triterpenoids belong to terpenoid compounds, are the largest class of plant secondary metabolites (Gershenzon and Dudareva, 2007). It has been generally believed that their physiological functions in plants are as part of plant defense systems, which enhances the resistance of plants against the invasion of plant pathogens and defend against environmental stress as well as provides repair mechanism for wounds and injuries (Domingo et al., 2009). Variety of infectious organisms were reported inhibited by triterpenoids compounds, including fungal, viral, bacterial, protozoal and parasites (Si et al., 2018).
Corresponding author. E-mail addresses: [email protected] (C. Shu), [email protected] (W. Jiang).
https://doi.org/10.1016/j.scienta.2019.108636 Received 26 May 2019; Received in revised form 1 July 2019; Accepted 2 July 2019 Available online 11 July 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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2.3. Effects of UA on A. alternata in vitro
Ursolic acid (3β-hyroxy-12-urs-2-en-28-oic acid, UA) is a pentacyclic triterpenoid widely distributed in the plant kingdom with various biological activities such as antioxidative, antitumor, anti-inflammatory, antiviral and antimicrobial activity (Jesus et al., 2015; López-Hortas et al., 2018). Ursolic acid is frequently isolated and identified closely associated with the potent antimicrobial activity of some medical plant extracts (Fontanay et al., 2008). It is also characterized as one of the central components of wax-like protective coatings of various fruits which provides resistance to fungal invasion in fruits (Cargnin and Gnoatto, 2017; Poirier et al., 2018). In recent years, the antifungal property of ursolic acid has been increasingly recognized and studied, previous researches have reported its antifungal activity against several postharvest pathogen species (Jabeen et al., 2011; Mahlo et al., 2013; Shaik et al., 2016). The findings from previous studies suggest that the carboxylic group at C-27 and the hydroxyl group at C-24 in this type of triterpenoid are critical for the rapid bactericidal activity (Zheng et al., 2008). Also, the high lipophilicity of UA was the necessary chemical structural features of its antimicrobial property, it may bind to the hydrophobic region of proteins and dissolve in lipid phases, which leads to membrane disturbance (Si et al., 2018). Additionally, a under field study conducted by Shaik et al. (2016) have shown that exogenous ursolic acid spray treatment would induce resistance response of sorghum grain which is associated with the induced salicylic acid production. Due to its low toxicity as well as the appreciable antifungal eff ;ects against postharvest pathogens, this has alerted us its application potential as a postharvest fungicide. However, the antifungal efficiency and the mode of actions of the antifungal property of ursolic acid remains unclear. More important, the inhibitory efficiency of ursolic acid against black spot rot and preserve apple fruit were rather limited. Therefore, this dissertation seeks to explain the antifungal efficiency of ursolic acid against A. alternata and its inhibitory effects of black spot rot on apple fruit. The main issues addressed in this paper are: (I) evaluate the in vitro and in vivo antifungal efficiency of ursolic acid against A. alternata; (II) explore the possible mode of actions of the antifungal property of UA; (III) evaluate the effect of UA treatment on the development of black spot rot and the induced-defense responses in apple fruit. The study offers some new insights into further ascertain its modes of action against the pathogen and would provide a theoretical basis for the better utilization of ursolic acid ahead to control postharvest Alternaria diseases in fruits.
The antifungal activity of UA in vitro was detected by measuring the mycelial radial growth and the spore germination. The radial hyphal growth was measured according to the procedure of Li et al. (2018a,b). A mycelial agar disk (5 mm in diameter) was divided from a 14-day-old colony then placed on the center of a 9 cm diameter petri dish containing 15 mL PDA with various final concentrations of UA at 0, 25, 50, 100, 200, 500, 800 and 1000 μg/mL. During the incubation at 28 °C, radial growth was measured daily by decussation method. The inhibition rate of UA was calculated based on the inhibit percentage of radial growth after 9 days of incubation. All of the experiments were performed in triplicates. The logarithm model was applied to describe the relationship between UA concentrations and inhibition rates. The inhibitory effects of UA on spore germination of A. alternata were tested as described previously (Pane et al., 2016) with some modifications. The spore suspension (1 × 106 spores/mL) was cultured in PDB and exposed to different concentrations of UA (0, 25, 50, 100, 200, 500, 800 and 1000 μg/mL). The treated culture was maintained at 28 °C in a shaking incubator at 160 rpm. Spore germinations were then assayed at 2, 4, 6, 8 and 12 h during inoculation. Spores were considered germinated when the length of a germ tube eclipsed half of the small-end diameter of the spore. The spore germination rates (%) were expressed as the percentage of germinated spores by the total spore number observed in the view fields. At least 200 spores were randomly observed. The experiment was repeated three times. All of the experiments were performed in triplicates. The logarithm model was applied to describe the relationship between UA concentrations and spore germination inhibition. 2.4. Morphology observation by scanning electronic microscopy (SEM)
2. Materials and methods
SEM observations were performed to check the morphology changes of A. alternata spores and mycelia incubated with UA. Preparation of samples was conducted according to the method described by (Li et al., 2018a,b) with some slight modifications. Briefly, spores and mycelial agar disks were added into 0 (control), 200 and 1000 μg/mL UA solution and incubated at 28 °C for 3 days. Then the samples were centrifuged (5000×g, 4 °C) for 10 min and washed twice with PBS buffer (100 mM, pH 7.4). After being dehydrated in ethanol and critical-pointdried with CO2 and coated by gold, the samples were examined with SEM (SU-8010, Hitachi, Tokyo, Japan) operating at 10 kV at 5000× level of magnification.
2.1. Chemicals
2.5. Assay of plasma membrane integrity with propidium iodide (PI) dyeing
Ursolic acid was purchased from Macklin Co. (Shanghai, China, purity > 99%). UA was dissolved in DMSO to prepare the stock solution and added to experimental solutions to obtain the required concentrations as described by previous publication (Mahlo and Eloff, 2014). All other reagents used in the experiments were of analytical grade.
Membrane integrity of A. alternata cells exposed to UA was evaluated by the previous method (Xu et al., 2018) with slight modifications. Spores and mycelia were incubated with 0 (control), 200 and 1000 μg/mL UA solution in a water-bath rotary shaker (120 rpm, 28 °C) for 12 h. Then the samples were centrifuged (5000×g, 4 °C) for 5 min and washed thrice with PBS buffer (50 mM, pH 7.2). Afterward, the samples were stained with 10 μg/mL PI at 30 °C for 5 min in the dark. Then the samples were centrifuged (5000×g, 4 °C) for 5 min and washed triple times with PBS to remove the remained dye and resuspended in the same PBS buffer. The samples were observed by confocal laser scanning microscopy (Fluoview FV1000 Espectral, Olympus, Tokyo, Japan) equipped with a digital camera at 546 nm excitation and 590 nm emission wavelengths. Three fields of view were randomly observed for each treatment and the experiment was repeated three times.
2.2. Preparation of fungal materials The pathogen Alternaria alternata (Fr.) Keissler was obtained from the microbiology lab of the China Agricultural University and maintained on potato dextrose agar (PDA) plates at 28 °C for 14 days. The spore suspension was obtained by flooding the plates with sterile distilled water and rubbing the surface of the colony gently with a sterile glass triangular rod. The concentration of the spore suspension was determined using a hemocytometer, with the density of 1 × 106 spores/ mL. The mycelia of A. alternata was obtained by adding an aliquot of 100 μL spore suspension into 20 mL of PDB medium and incubated at 28 °C for 3 days, the fungal broth was abandoned and the mycelia pellet was collected, then washed twice with sterile distilled water for further use (Yuan et al.. 2018).
2.6. Cytoplasmic content leakage detection The permeability of A. alternata cell membranes was indicated by their electric conductivity and was detected as described previously 2
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described in the publication (Izard and Limberger, 2003). A standard calibration curve using cholesterol as standard was applied to determine total lipids contents. The data were expressed as mg/g dry weight of mycelia.
(Wang et al., 2015) with some modifications. The culture of mycelium was the same as Section 2.2. The sample of 0.3 g mycelia were suspended in 20 mL of solutions containing UA at 0 (control), 200 and 1000 μg/mL. The extracellular electric conductivity was determined continuously at 0, 0.5, 1, 2, 3 and 4 h by a conductivity meter. The results were indicated by extracellular conductivity values (μS/cm). The measurement of cytoplasmic content leakage of A. alternata mycelium was determined by previous methods described by (Li et al., 2018a, b) with minor modifications. The mycelial clumps were suspended in 40 mL solutions containing 0, 200, 1000 μg/mL UA to incubate for 4 h. During the incubation, aliquots 5 mL of the aqueous solution were sampled each hour to determine the cytoplasmic content leakage. Microporous membranes (0.22 μm) were applied to remove floating mycelium and fragments and the filtrate was collected for further determinations. Soluble protein contents were determined according to the Bradford assay, bovine serum albumin (BSA) was applied as the standard to quantified the release of protein. Soluble sugar contents were measured by the phenol-sulfuric acid method, glucose was used as the standard to quantify the soluble sugar values. Nucleotide content was measured by detecting the absorbance values at 260 nm by an UV/Vis spectrophotometer. All of the experiments were performed in triplicates.
2.8. Effects of UA treatment on controlling black spot rot in apple fruit The antifungal efficiency of UA treatment on black spot rot development on apple fruit was conducted based on the method reported previously with slight modifications (Ge et al., 2019b; Ji et al., 2018). Apple fruits (cv. Fuji) were harvested at commercial maturity from an orchard in Beijing and transported to the laboratory immediately. The fruits have no pathogen infections and physical injuries. The surface of fruits was disinfected with 1.0% (v/v) sodium hypochlorite for 1 min and completely washed with distilled water, then air-dried at room temperature before further use. Then the fruits were divided into two groups, (I) fruits were directly inoculated after air dried, then 10 μL of spore suspension at the concentration of 1 × 106 /mL were injected into the uniform size wounds (5 mm deep, 2 mm wide) at equator positions of the fruit. The fruits were air-dried for 1 h, then equal volumes of UA solutions at 0 (distilled water set as control), 200 and 1000 μg/ mL were injected into the same wounds; (II) fruits were dipped in 0 and 200 μg/mL UA solution for 10 min, air-dried again. Aliquot of 10 μL spore suspension at the concentration of 1 × 106 /mL was injected into uniform size wounds (5 mm deep, 2 mm wide) at equator positions of the fruit. Finally, all the fruits were stored in clean boxes maintaining a humidity of 85–90%, and stored at 20 °C. Lesion diameter reflects the severity of the disease for each treatment. Each treatment included three replicates of 30 fruits each and the experiment was performed thrice.
2.7. Determination of oxidative stress induced by UA Spores and mycelia were treated with the same method described in Section 2.5. Then the treated fungus was stained with 10 μM DCFH-DA in the dark at 28 °C for 4 h. Then the mixture was centrifuged (5000×g, 4 °C) for 5 min and washed three times with PBS, and resuspended with the same buffer again. To demonstrate the ROS production is directly induced by the antifungal effect of UA, the change of fluorescence intensity of the sample in the presence of the antioxidant L-cysteine (5 mM, 28 °C) was investigated after washing off the fluorescent stain (Helmerhorst et al., 2001). The fluorescence intensity was observed by confocal laser scanning microscopy and quantitative determinate by a spectrofluorometer (Shimadzu RF-5301PC, Shimadzu, Kyoto, Japan) at an excitation wavelength of 488 nm and an emission wavelength of 529 nm. Three fields of view were randomly chosen for each treatment, and the experiment was repeated three times. The MDA content was determined as the method described previously (Wang et al., 2013) with some modifications. The mycelia were suspended in 20 mL solution containing different concentrations of UA (0, 200 and 1000 μg/mL) and incubated on a water-bath rotary shaker at 28 °C for 24 h. Sample of 1.0 g treated mycelia were homogenized with 5 mL of pre-cooled sodium phosphate buffer (100 mM, pH 7.8) mix with quartz sand. After centrifuged for 20 min (12,000 × g, 4 °C), the supernatants were collected for MDA determination. The content of MDA was tested by the thiobarbituric acid reactive substances assay, the absorbance was measured at the wavelength of 532 nm and 600 nm. The concentration was expressed as μmol/g fresh weight mycelial. Total lipid contents of A. alternata were determined with the phosphovanillin method described by (Xu et al., 2018) with some modifications. The culture and treatment of the mycelia were with the same method described in Section 2.7, then the treated mycelia were vacuum freeze-dried for 8 h. Following that a known weight of dry mycelia was homogenized with 4.0 mL of extract solution containing methanol: chloroform: water mixture (2:1:0.8, v/v/v), the mixture was dramatically shaking for extracting lipids. The samples were centrifuged at 4000 × g for 10 min, and the lower phase was taken to mix with 0.2 mL of saturated sodium chloride solution. The lipid extraction was mixed with 0.2 mL of chloroform, transferred to a new tube and 0.5 mL of H2SO4 was added, heated in a boiling water bath for 10 min and left to cool at room temperature. An aliquot of 5.0 mL of phosphoric acid-vanillin reagent was added to each tube and incubated for 15 min at 37 °C. The absorbance at 520 nm was detected. The details for the preparation of the phosphoric acid-vanillin reagent are the same as
2.9. Determination of UA treatment on quality parameters of apple fruit and defense-related enzyme activities The quality parameters of infected apple fruit were measured referred to the previous study (Ge et al., 2019b). The pulp firmness was measured using a force gauge with a 0.1–2 cm diameter flat probe (Zhejiang Tuopu Instrument Co., Ltd.). Soluble solids contents (SSC) were measured with a digital refractometer (PAL-1, Atago, Tokyo, Japan). Titratable acidity (TA) was determined by acid-base titration method. The result was expressed as the percentage of malic acid. Activities of PAL and PPO were measured by the spectroscopy methods (Ge et al., 2019b; Jiao et al., 2018). The enzyme activities were calculated as the change in U/g, taking one unit of enzyme activity as equal to 0.01 ΔOD per min. 2.10. Statistical analysis All the experiments were completed by a randomized design and done in triplicate; each replicate included three induvial determinations. Data were performed with SPSS software (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) and Duncan's multi-range test were performed to determine significant differences between the means, where the data were considered statistically significantly when p < 0.05. 3. Results 3.1. In vitro antifungal activity of UA against A. alternata UA significantly (p < 0.05) inhibited the extension of colonies and the inhibition exerted a dose-dependent manner (Fig. 1A). As the increasing concentration of UA, the colony diameters decreased significantly. When the concentration reached 200 μg/mL, the mycelial growth was inhibited by 45.6%, whereas 1000 μg/mL UA treatment roughly inhibited the mycelial growth, reached an inhibition by 70.1%. 3
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A logarithm model was used to describe the relationship between UA concentrations and inhibition rates after 9 days of incubation. The halfinhibition concentration of UA was calculated to 284.5 μg/mL, the R2 is 0.9914, indicating a good logarithmic relationship between concentrations and inhibition rates (Fig. 1A). The presence of UA inhibited spore germinations as well (Fig. 1B). The result showed that spore germination was significantly retarded (p < 0.05) exposed to UA. The spore germination rate is 82.7% in the control, whereas it was significantly inhibited by 200 μg/mL UA. The highest UA concentration (1000 μg/mL) prompted the most profound reduction of spore germination, the inhibition of 67.9% was detected. These results indicating that UA exhibited significant antifungal efficiency against A. alternata in vitro. 3.2. Effects of UA treatment on morphology changes of A. alternata Morphology alternations of spores and mycelial were observed by SEM micrographs. The spores in the control showed normal morphology of mature spores with obclavate to obpyriform structure with longitudinal and transverse septa and germ tube and warts dispersed on the surface (Fig. 2A), whereas the surface morphology of treated spores was irreversible altered after incubation exposed to 200 μg/mL UA (Fig. 2B–C). The treated spores showed significantly shriveled and recessed. Greater concavity and major broke on the surface could be observed exposed to 1000 μg/mL UA (Fig. 2C). Mycelia in the control showed the typical morphology with uniform, sturdy, thick and linearly shape (Fig. 2D), while hyphae emerged distorted and crumbled during incubation exposed to 200 mg/L UA (Fig. 2E). Seriously collapsed squashed and loss of linearity occurred in the mycelia incubated with 1000 μg/mL UA (Fig. 2F). Fig. 1. Effect of ursolic acid (UA) concentrations on in vitro growth of A. alternata. Effects of UA on colony diameter and its inhibition rate after incubation at 28 °C for 9 days (A). Effects of UA on spore germination after incubation at 28 °C for 12 h (B). The logarithm model was applied to describe the relationship between UA concentrations and inhibition rates. Untreated spores served as the control. Each value is the mean of three replicates.
3.3. Effects of UA on plasma membrane integrity, permeability and cellular leakage of A. alternata The integrity of the plasma membrane was detected qualitatively by laser confocal microscopy by PI straining. Both spores and mycelium of A. alternata grown exposed to UA showed strong red fluorescence, and the fluorescence intensity showed a dose effect (Fig. 3A). Whereas no
Fig. 2. Scanning electron microscopy images of spores (A–C) and mycelia (D–F) of A. alternata exposed to different concentrations of UA treatment. Normal morphology of spores was with the obclavate to obpyriform structure with longitudinal and transverse septa and germ tube and warts dispersed on the surface. Normal mycelia were with uniform, sturdy, thick and linearly shape. Lesioned cells showed distortion, sunken and shriveled morphology. Untreated spores and mycelia were served as the control. 4
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Fig. 3. Effects of ursolic acid on membrane integrity of A. alternata. Confocal laser scanning microscopy images showing the loss of cell plasma membrane integrity of A. alternata exposed to UA at different concentrations (A), cells with damaged plasma membranes exhibit red fluorescence. Effects of different concentrations of UA treatment on cytoplasmic leakage. Extracellular conductivity (B), proteins (C), sugars (D), nucleic acids (E) of A. alternata treated with UA. Untreated spores and mycelia were served as the control. Each value is the mean of three replicates. The vertical bars represent the standard deviations of the means.
obvious fluorescence was observed in the control demonstrating high cell membrane integrity. These results showed that UA may cause damages and alterations to cell membrane integrity thus increases the permeability and induces the debilitation or even death of the fungi. Electric conductivity value represented the permeability of A. alternata cell membranes. There was an immediate dramatic increase in extracellular conductivity detected when A. alternata were exposed to
UA at various concentrations, as well as with prolonged exposure time (Fig. 3B). With the concentration of UA was 200 μg/mL, conductivity values increased significantly (p < 0.05) after 30 min compared to the control (79.3 ± 0.98 μS/cm). The extracellular conductivity of A. alternata in the control was slightly increased, whereas it was increased steadily during the incubation when exposed to UA. At the end of incubation, the conductivity value was increased by 17.75% (p < 0.05) 5
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Fig. 4. Effects of ursolic acid on induced oxidative stress of A. alternata. Confocal laser scanning microscopy images showing the accumulation of cytosolic reactive oxygen species (ROS) of A. alternata exposed to UA at different concentrations (A), spores and mycelial with intracellular ROS induction exhibit green fluorescence. Effects of different concentrations of UA treatment on endogenous ROS production (B), lipid peroxidation (C) and total membrane lipid content (D) of A. alternata. Untreated spores and mycelia were served as the control. Each value is the mean for three replicates. The vertical bar indicates the standard deviation. Different letters indicate significant differences among sample groups (ANOVA, P < 0.05).
and 32.6% (p < 0.05) by the exposure to UA at 200 and 1000 μg/mL, respectively. The leakage of cytoplasmic contents was further determined to investigate the alteration in membrane integrity of A. alternata. The results indicated that UA induced significant leakages of soluble protein, soluble sugar and nucleic acids from A. alternata (Fig. 3C–E). The leakage of cytoplasmic contents increased rapidly after 1 h of incubation and remained increase gradually during the subsequent incubation, and the degree of leakage were positively correlated with UA concentration. These results indicated that UA induced significant damages and alterations to the cell membrane, causing leakages of cytoplasmic
content of A. alternata.
3.4. UA induced intracellular ROS production and lipid peroxidation The distribution of intracellular ROS in spores and mycelial was observed by confocal laser scanning microscopy (CLSM). As showed in Fig. 4A, both spores and mycelia cells of A. alternata grown exposed to UA showed visible green fluorescence, indicating that the accumulation of intracellular ROS, while no noticeable fluorescence was observed in the cells grown in the control. An obvious increase of fluorescence intensity observed in the cells incubated with 200 μg/mL UA. More 6
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exposure to 200 and 1000 μg/mL UA compared to the control (Fig. 4B). Antioxidant L-cysteine was used to further verification the oxidative stress effect of conidia induced by UA. As shown in Fig. 4B, the fluorescence signals were significantly quenched (p < 0.05) after the addition of L-cysteine, and maintain in a relatively low level, indicating that the increasements of fluorescence induced by UA were the result of endogenous ROS formation and accumulation. MDA can be determined as the indicator of oxidative damage of the fungal plasma membrane and could further confirm the results of the ROS assay. Compared to the control, the MDA content of the fungi exposed to 200 and 1000 μg/mL UA treatment was increased by 85.63% and 1.16-times, which demonstrating that UA treatment induced severe lipid peroxidation and caused severe lipid peroxidation in A. alternata. On the other hand, the total lipids content in A. alternata were significantly reduced (p < 0.05) by incubation exposed to UA treatments (Fig. 4D), the total lipids content in control is 19.75% and 44.52% higher compared to 200 and 1000 μg/mL UA treatment respectively, which consistent with the trend of ROS and MDA content. 3.5. Efficacy of UA in controlling black spot rot in apple fruit The development of black spot lesions in apple fruit was significantly (p < 0.05) suppressed by UA treatments (Fig. 5A). After incubation for 3 days, UA treatments significantly (p < 0.05) suppressed the startup of the disease, and on the 12th day after inoculation, lesion diameter of UA treatment group decreased by 18.76% and 34.47% respectively when compared to control. This demonstrated that UA exerts direct inhibitory effect on A. alternata, thus would inhibit the occurrence and suppress the development of black spot rot on apple fruits. In addition, UA dipping treatment could also inhibit the development of lesion, the lesion diameter was 14.95% smaller than control fruit for 12 days after inoculation. These results suggested that apart from the direct antifungal activity, UA could induce the defense responses in apple fruit against postharvest black spot rot caused by A. alternata. Notably, no surface injuries or phytotoxicity was observed in the fruit even treated with the highest concentration of UA. Also, there were no significant differences in postharvest quality parameters, including firmness, SSC, pH and TA of apple fruit among all treatments during 12 days of storage (Table 1). PAL and POD are important defense enzymes. The PAL activities in apple fruits dipping with UA were 54.51% and 11.92% higher (p < 0.05) than that in control respectively, after 3 and 6 days of storage (Fig. 5B). The activity of POD increased rapidly in fruits treated with UA and was significantly (p < 0.05) higher than that of control during storage after 3 days and the subsequent, than that of control (Fig. 5C). This indicated that UA treatment significantly activated PAL and POD enzyme activities during storage, which play an important role in induced resistance of fruit. 4. Discussion Fig. 5. Effect of UA treatments on lesion diameter of black spot rot on apple fruit inoculated with A. alternata. Effect of UA treatment (A) on lesion diameter development of apple fruit. Effect of UA immersion treatment on PAL (B) and PPO (C) activities of apple fruit. Fruits treated with distilled water were served as the control. Each value is the mean of three replicates. The vertical bars represent the standard deviations of the means. Different letters indicate significant differences among sample groups (ANOVA, P < 0.05).
Apple fruit is one of the most important fruit crops worldwide, however, postharvest decay by pathogen fungi lead to tremendous economic losses. The present work investigated the antifungal efficiency of ursolic acid, a natural triterpenoid, against A. alternata and its possible mechanism involved as well as the potential of UA to preserve apple fruit was further evaluated. We found that the colony growth and spore germination of A. alternata, as well as black spot lesions in apple fruits, could be significantly inhibited by UA treatment. The physiological function of the triterpenoid compound is to defend against plant pathogens invasion (Domingo et al., 2009). UA is the main antifungal component isolated from some medical herbs such as Rosmarinus officinalis (Collins and Charles, 1987), Breonadia salicina (Mahlo and Eloff, 2014) and Swertia chirata (Kaur et al., 2019). Previous studies have also reported the antifungal activity of UA against several postharvest fungal
intense and steady green fluorescence was observed in the cells incubated with 1000 μg/mL UA. This delegated that induced intracellular ROS generation and accumulation in the spores and mycelium of A. alternata with UA exposure. The intracellular ROS produced in the spores was determined quantitatively by fluorescence spectrophotometer. The fluorescence strength in spores was increased by 86.3% and 2.38-fold by the 7
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Table 1 Effects of UA treatment on quality parameters of apple fruit during storage at 25 °C for 12 days. Treatment
Firmness (kg/cm2)
SSC (%)
Initial UA 200 μg/mL injection UA 1000 μg/mL injection UA 200 μg/mL dipping
13.21 11.38 11.37 12.12
14.76 14.87 14.62 14.23
± ± ± ±
0.37a 0.42b 0.45b 0.32ab
± ± ± ±
pH 0.33a 0.26a 0.34a 0.35a
3.25 3.68 3.65 3.57
TA (%) ± ± ± ±
0.14a 0.12b 0.13b 0.13b
0.398 0.393 0.384 0.386
± ± ± ±
0.04a 0.04a 0.03b 0.02b
Apple fruit were divided into two groups, (I) fruits were injected with 10 μL of UA solutions at 200 and 1000 μg/mL at the wounds (5 mm deep, 2 mm wide) at equator positions of the fruit, distilled water set as control; (II) fruits were immersed with UA at 0 and 200 μg/mL for 10 min at room temperature. Firmness, soluble solids contents, pH values, and titratable acidity of apple fruit were measured on the 12th day of storage at 25 ℃. Each value is represented as mean of three replicates ± standard deviation. Values followed by different letters are significant different according to Duncan’s multiple range test (P < 0.05).
postharvest Alternaria rot on fruits. Further, its effects on the defense system of fruits were investigated. Our results showed that UA immersion treatment would suppress the development of black spot rot on apple fruit and the activation activity of PAL and POD were detected. PAL is a key enzyme in the metabolism of plant phenylpropanoid, which is essential for the biosynthesis of secondary metabolites and plays an important role in the normal growth and development of plants and defense against pathogens (Jiao et al., 2018). POD is associated with the strengthening of plant cell walls by catalyzing lignin biosynthesis, which act as an effective barrier against pathogen penetration and spread (Kärkönen and Koutaniemi, 2010). The enhanced PAL and POD activity is associated with the increase of resistance system, such as in apple (Ge et al., 2019a), peach (Jiao et al., 2018) and pear (Yu et al., 2014) fruit. A study by Shaik et al. (2016) also reported the exogenous ursolic acid spray treatment would inhibit sorghum grain mold by inducing salicylic acid production, which is associated with the enhanced plant resistance system. These findings suggest that UA could induced the defense responses in apple fruit against the postharvest A. alternata invasion. The existence of reports on the synthesis of UA reveals the scope of its commercialization (López-Hortas et al., 2018) encouraging us to study the wide range of application in the future. Additionally, further studies should be made to dissect the antifungal membrane binding sites of UA in more detail and its molecular mechanisms underlying in more details.
species (Jabeen et al., 2011; Mahlo and Eloff, 2014; Shaik et al., 2016). Our results suggest that UA exerts in vitro antifungal activity and reduce disease development in apple fruit. The plasma membrane is an important target for antifungals. Considering the lipophilic character of UA, we investigated whether UA interacts with the cell membrane of A. alternata. The morphology observation indicated the prominent damage on spores and mycelial cell wall after UA treatment, which suggested that UA may induced membrane disturbance resulted in disruption of the cell wall and lysis of the cell. UA may preferentially partition from the aqueous phase and penetrate and accumulate in the phospholipid bilayer, which would damage the phospholipids layers and induces the decreased vitality of the cell (da Cruz Cabral et al., 2013). The spores and mycelia treated by UA could be stained with PI and the fluorescence intensity showed a dose effect, which indicated the lesioned membrane integrity. Our result was in line with Xu et al (2018) who found an increased influx of PI into A. alternata exposed to cinnamaldehyde, a hydrophobic monoterpene compound. Meanwhile, was detected a significant leakage of cytoplasmic content of A. alternata treated with UA in this study. The lipophilic property and hydrophobic scaffold of UA could bind to the hydrophobic region of proteins and dissolve in lipid phases, leading the impairment of membrane-bound proteins which maintain homeostasis in the plasma membrane (Si et al., 2018). The cytoplasmic content of A. alternata may leak into the extracellular fluid due to the damage of cell membrane during growth. Ji et al. (2018) reported that methyl thujate treatment increased the intracellular macromolecular cytoplasmic components leakage, which is associated with the damaged cell membrane integrity. These results suggest that UA exerts antifungal activity via the membrane-targeted mechanism. Disruption of membrane function and integrity due to ROS oxidation of membrane lipids is part of some of the antifungal potent modes of action (Rautenbach et al., 2016). The current study indicated that the fluorescence intensity of ROS was enhanced obviously in the treated spores and mycelia, and it was positively correlated with the concentration of UA. In addition, the enhanced fluorescence intensity was quenched in the presence of the oxygen scavenger L-cysteine, which indicated the increase of fluorescence was indeed because of the ROS formation that contributes to contributes to its antifungal efficiency (Helmerhorst et al., 2001). Our results were consisting with the previous study which reported that UA treatment and plant extracts enriched in UA could induce ROS production in microorganisms (Oloyede et al., 2017). Redundant ROS would cause lipid peroxidation by attacking unsaturated fatty acids distributed in lipids. MDA is the hallmark product of lipid peroxidation, the MDA content increased significantly in UA treated group, suggesting UA induced serious lipid peroxidation and massive oxidative damage to the membrane occurred in A. alternata. Correspondingly, the result was supported by the decreased content of membrane lipids. Our results suggest that UA exerts directly antifungal activity through membrane damage and oxidative stress against A. alternata which ultimately lead to the collapse and death of the cells. The directly antifungal activity of UA on A. alternata revealed that it could be potentially used as an alternative fungicide for controlling
5. Conclusion In summary, ursolic acid could effectively inhibit A. alternata growth by the mode of membrane-targeted mechanism. UA induced the disturbance of membrane permeability, disruption of membrane integrity, and intracellular ROS accumulation in A. alternata result in the lysis of the pathogen, thus exert directly antifungal activity. In addition to its direct antifungal activity, UA induced the defense responses in apple fruit against postharvest black spot rot caused by A. alternata. UA is a triterpenoid widely distributed in many plant species, which suggest that application of UA may provide a novel effective strategy to control postharvest diseases of fruits. Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work. Acknowledgement This research was supported by the national key research and development projects of the China Ministry of Science and Technology (No. 2018YFD0401302). References Cargnin, S.T., Gnoatto, S.B., 2017. Ursolic acid from apple pomace and traditional plants:
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Antifungal efficacy of ursolic acid in control of Alternaria alternata causing black spot rot on apple fruit and possible mechanisms involved Chang Shu, Handong Zhao, Wenxiao Jiao, Bangdi Liu, Jiankang Cao, Weibo Jiang
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College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Qinghuadonglu Road, Beijing 100083, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Ursolic acid Black spot rot Apple fruit Alternaria alternata Antifungal activity
Black spot rot caused by Alternaria alternata is becoming a main postharvest disease on apple fruit. The objective of this study was to elucidate the antifungal efficiency of ursolic acid (UA) against A. alternata and its application in preserving apple fruit. The results showed that UA induced disturbance of membrane permeability and integrity, as well as intracellular ROS accumulation in A. alternata, result in the sabotaged morphology and lysis of the pathogen, thus exert directly antifungal activity. In addition, the application of UA significantly inhibited the black spot rot development on apple fruit, the increased defense enzyme activities were detected. These results suggested that, in addition to its direct antifungal activity, UA induced the defense responses in apple fruit against the postharvest pathogen invasion. UA is widely distributed in the plant kingdom and could be a promising alternative for Alternata diseases management.
1. Introduction Apple (Malus domestica Borkh.) fruit is one of the most cultivated and consumed fruit crops worldwide. The recent statistics from FAO (2017) showed that China is the largest apple producer. Apple fruits are mostly preserved in cold condition for long-term preserving and marketing (Shen et al., 2018). However, despite modern storage facilities, postharvest losses are still ubiquitous. Fungal pathogens are mainly responsible for significant economic losses. Black spot rot caused by Alternaria alternata is becoming an important postharvest disease on apple fruit in China since the widespread implementation of the practice of bagging apples during the period of fruit development (Dang et al., 2018; Ge et al., 2019a). This may be attributed to the higher temperature and humidity inside the bag, which made pathogens more possible to grow. A. alternata is a latent fungus that could contaminate the fruits through breaks in the skin or other weakened areas in the orchard and during the storage (Jijakli and Lepoivre, 2006) and could rapidly develop at low temperatures and cause tremendous postharvest losses during storage period and shelf life (Yan et al., 2015). Meanwhile, A. alternata can yield secondary metabolites considered as both phytotoxins and mycotoxins, which can be detrimental to humans, causing food safety issues (Wang et al., 2017). The persistent application of synthetic fungicides such as iprodione, tebuconazole, and mancozeb has gradually induced the emergence of fungicide-resistant strains as well as the environmental contamination, while the public
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has begun to concern more about the harmful effects of synthetic fungicides on human health and related negative effects (Yuan et al., 2019). Therefore, there is an urgent need to develop an effective and environmentally friendly alternative to these traditional fungicides to control black spot rot on apple fruit. The use of natural botanical ingredients is thought to be a good viable alternative to control postharvest diseases. Plant metabolites have long been used as preservatives in food preservation and traditional medicine (da Rocha Neto et al., 2019d), it has been reported that essential oils (Castro et al., 2017; Xu et al., 2018), phenolic acids and its derivatives (Li et al., 2018a,b; Shaik et al., 2016) as well as terpenoids (Jabeen et al., 2011; Mahlo et al., 2013; Mahlo and Eloff, 2014) could inhibit the growth of A. alternata and other postharvest pathogens and would be applied as natural fungicide for control postharvest decay. Among the compounds studied above, triterpenoids have attracted the attention among researchers. Triterpenoids belong to terpenoid compounds, are the largest class of plant secondary metabolites (Gershenzon and Dudareva, 2007). It has been generally believed that their physiological functions in plants are as part of plant defense systems, which enhances the resistance of plants against the invasion of plant pathogens and defend against environmental stress as well as provides repair mechanism for wounds and injuries (Domingo et al., 2009). Variety of infectious organisms were reported inhibited by triterpenoids compounds, including fungal, viral, bacterial, protozoal and parasites (Si et al., 2018).
Corresponding author. E-mail addresses: [email protected] (C. Shu), [email protected] (W. Jiang).
https://doi.org/10.1016/j.scienta.2019.108636 Received 26 May 2019; Received in revised form 1 July 2019; Accepted 2 July 2019 Available online 11 July 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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2.3. Effects of UA on A. alternata in vitro
Ursolic acid (3β-hyroxy-12-urs-2-en-28-oic acid, UA) is a pentacyclic triterpenoid widely distributed in the plant kingdom with various biological activities such as antioxidative, antitumor, anti-inflammatory, antiviral and antimicrobial activity (Jesus et al., 2015; López-Hortas et al., 2018). Ursolic acid is frequently isolated and identified closely associated with the potent antimicrobial activity of some medical plant extracts (Fontanay et al., 2008). It is also characterized as one of the central components of wax-like protective coatings of various fruits which provides resistance to fungal invasion in fruits (Cargnin and Gnoatto, 2017; Poirier et al., 2018). In recent years, the antifungal property of ursolic acid has been increasingly recognized and studied, previous researches have reported its antifungal activity against several postharvest pathogen species (Jabeen et al., 2011; Mahlo et al., 2013; Shaik et al., 2016). The findings from previous studies suggest that the carboxylic group at C-27 and the hydroxyl group at C-24 in this type of triterpenoid are critical for the rapid bactericidal activity (Zheng et al., 2008). Also, the high lipophilicity of UA was the necessary chemical structural features of its antimicrobial property, it may bind to the hydrophobic region of proteins and dissolve in lipid phases, which leads to membrane disturbance (Si et al., 2018). Additionally, a under field study conducted by Shaik et al. (2016) have shown that exogenous ursolic acid spray treatment would induce resistance response of sorghum grain which is associated with the induced salicylic acid production. Due to its low toxicity as well as the appreciable antifungal eff ;ects against postharvest pathogens, this has alerted us its application potential as a postharvest fungicide. However, the antifungal efficiency and the mode of actions of the antifungal property of ursolic acid remains unclear. More important, the inhibitory efficiency of ursolic acid against black spot rot and preserve apple fruit were rather limited. Therefore, this dissertation seeks to explain the antifungal efficiency of ursolic acid against A. alternata and its inhibitory effects of black spot rot on apple fruit. The main issues addressed in this paper are: (I) evaluate the in vitro and in vivo antifungal efficiency of ursolic acid against A. alternata; (II) explore the possible mode of actions of the antifungal property of UA; (III) evaluate the effect of UA treatment on the development of black spot rot and the induced-defense responses in apple fruit. The study offers some new insights into further ascertain its modes of action against the pathogen and would provide a theoretical basis for the better utilization of ursolic acid ahead to control postharvest Alternaria diseases in fruits.
The antifungal activity of UA in vitro was detected by measuring the mycelial radial growth and the spore germination. The radial hyphal growth was measured according to the procedure of Li et al. (2018a,b). A mycelial agar disk (5 mm in diameter) was divided from a 14-day-old colony then placed on the center of a 9 cm diameter petri dish containing 15 mL PDA with various final concentrations of UA at 0, 25, 50, 100, 200, 500, 800 and 1000 μg/mL. During the incubation at 28 °C, radial growth was measured daily by decussation method. The inhibition rate of UA was calculated based on the inhibit percentage of radial growth after 9 days of incubation. All of the experiments were performed in triplicates. The logarithm model was applied to describe the relationship between UA concentrations and inhibition rates. The inhibitory effects of UA on spore germination of A. alternata were tested as described previously (Pane et al., 2016) with some modifications. The spore suspension (1 × 106 spores/mL) was cultured in PDB and exposed to different concentrations of UA (0, 25, 50, 100, 200, 500, 800 and 1000 μg/mL). The treated culture was maintained at 28 °C in a shaking incubator at 160 rpm. Spore germinations were then assayed at 2, 4, 6, 8 and 12 h during inoculation. Spores were considered germinated when the length of a germ tube eclipsed half of the small-end diameter of the spore. The spore germination rates (%) were expressed as the percentage of germinated spores by the total spore number observed in the view fields. At least 200 spores were randomly observed. The experiment was repeated three times. All of the experiments were performed in triplicates. The logarithm model was applied to describe the relationship between UA concentrations and spore germination inhibition. 2.4. Morphology observation by scanning electronic microscopy (SEM)
2. Materials and methods
SEM observations were performed to check the morphology changes of A. alternata spores and mycelia incubated with UA. Preparation of samples was conducted according to the method described by (Li et al., 2018a,b) with some slight modifications. Briefly, spores and mycelial agar disks were added into 0 (control), 200 and 1000 μg/mL UA solution and incubated at 28 °C for 3 days. Then the samples were centrifuged (5000×g, 4 °C) for 10 min and washed twice with PBS buffer (100 mM, pH 7.4). After being dehydrated in ethanol and critical-pointdried with CO2 and coated by gold, the samples were examined with SEM (SU-8010, Hitachi, Tokyo, Japan) operating at 10 kV at 5000× level of magnification.
2.1. Chemicals
2.5. Assay of plasma membrane integrity with propidium iodide (PI) dyeing
Ursolic acid was purchased from Macklin Co. (Shanghai, China, purity > 99%). UA was dissolved in DMSO to prepare the stock solution and added to experimental solutions to obtain the required concentrations as described by previous publication (Mahlo and Eloff, 2014). All other reagents used in the experiments were of analytical grade.
Membrane integrity of A. alternata cells exposed to UA was evaluated by the previous method (Xu et al., 2018) with slight modifications. Spores and mycelia were incubated with 0 (control), 200 and 1000 μg/mL UA solution in a water-bath rotary shaker (120 rpm, 28 °C) for 12 h. Then the samples were centrifuged (5000×g, 4 °C) for 5 min and washed thrice with PBS buffer (50 mM, pH 7.2). Afterward, the samples were stained with 10 μg/mL PI at 30 °C for 5 min in the dark. Then the samples were centrifuged (5000×g, 4 °C) for 5 min and washed triple times with PBS to remove the remained dye and resuspended in the same PBS buffer. The samples were observed by confocal laser scanning microscopy (Fluoview FV1000 Espectral, Olympus, Tokyo, Japan) equipped with a digital camera at 546 nm excitation and 590 nm emission wavelengths. Three fields of view were randomly observed for each treatment and the experiment was repeated three times.
2.2. Preparation of fungal materials The pathogen Alternaria alternata (Fr.) Keissler was obtained from the microbiology lab of the China Agricultural University and maintained on potato dextrose agar (PDA) plates at 28 °C for 14 days. The spore suspension was obtained by flooding the plates with sterile distilled water and rubbing the surface of the colony gently with a sterile glass triangular rod. The concentration of the spore suspension was determined using a hemocytometer, with the density of 1 × 106 spores/ mL. The mycelia of A. alternata was obtained by adding an aliquot of 100 μL spore suspension into 20 mL of PDB medium and incubated at 28 °C for 3 days, the fungal broth was abandoned and the mycelia pellet was collected, then washed twice with sterile distilled water for further use (Yuan et al.. 2018).
2.6. Cytoplasmic content leakage detection The permeability of A. alternata cell membranes was indicated by their electric conductivity and was detected as described previously 2
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described in the publication (Izard and Limberger, 2003). A standard calibration curve using cholesterol as standard was applied to determine total lipids contents. The data were expressed as mg/g dry weight of mycelia.
(Wang et al., 2015) with some modifications. The culture of mycelium was the same as Section 2.2. The sample of 0.3 g mycelia were suspended in 20 mL of solutions containing UA at 0 (control), 200 and 1000 μg/mL. The extracellular electric conductivity was determined continuously at 0, 0.5, 1, 2, 3 and 4 h by a conductivity meter. The results were indicated by extracellular conductivity values (μS/cm). The measurement of cytoplasmic content leakage of A. alternata mycelium was determined by previous methods described by (Li et al., 2018a, b) with minor modifications. The mycelial clumps were suspended in 40 mL solutions containing 0, 200, 1000 μg/mL UA to incubate for 4 h. During the incubation, aliquots 5 mL of the aqueous solution were sampled each hour to determine the cytoplasmic content leakage. Microporous membranes (0.22 μm) were applied to remove floating mycelium and fragments and the filtrate was collected for further determinations. Soluble protein contents were determined according to the Bradford assay, bovine serum albumin (BSA) was applied as the standard to quantified the release of protein. Soluble sugar contents were measured by the phenol-sulfuric acid method, glucose was used as the standard to quantify the soluble sugar values. Nucleotide content was measured by detecting the absorbance values at 260 nm by an UV/Vis spectrophotometer. All of the experiments were performed in triplicates.
2.8. Effects of UA treatment on controlling black spot rot in apple fruit The antifungal efficiency of UA treatment on black spot rot development on apple fruit was conducted based on the method reported previously with slight modifications (Ge et al., 2019b; Ji et al., 2018). Apple fruits (cv. Fuji) were harvested at commercial maturity from an orchard in Beijing and transported to the laboratory immediately. The fruits have no pathogen infections and physical injuries. The surface of fruits was disinfected with 1.0% (v/v) sodium hypochlorite for 1 min and completely washed with distilled water, then air-dried at room temperature before further use. Then the fruits were divided into two groups, (I) fruits were directly inoculated after air dried, then 10 μL of spore suspension at the concentration of 1 × 106 /mL were injected into the uniform size wounds (5 mm deep, 2 mm wide) at equator positions of the fruit. The fruits were air-dried for 1 h, then equal volumes of UA solutions at 0 (distilled water set as control), 200 and 1000 μg/ mL were injected into the same wounds; (II) fruits were dipped in 0 and 200 μg/mL UA solution for 10 min, air-dried again. Aliquot of 10 μL spore suspension at the concentration of 1 × 106 /mL was injected into uniform size wounds (5 mm deep, 2 mm wide) at equator positions of the fruit. Finally, all the fruits were stored in clean boxes maintaining a humidity of 85–90%, and stored at 20 °C. Lesion diameter reflects the severity of the disease for each treatment. Each treatment included three replicates of 30 fruits each and the experiment was performed thrice.
2.7. Determination of oxidative stress induced by UA Spores and mycelia were treated with the same method described in Section 2.5. Then the treated fungus was stained with 10 μM DCFH-DA in the dark at 28 °C for 4 h. Then the mixture was centrifuged (5000×g, 4 °C) for 5 min and washed three times with PBS, and resuspended with the same buffer again. To demonstrate the ROS production is directly induced by the antifungal effect of UA, the change of fluorescence intensity of the sample in the presence of the antioxidant L-cysteine (5 mM, 28 °C) was investigated after washing off the fluorescent stain (Helmerhorst et al., 2001). The fluorescence intensity was observed by confocal laser scanning microscopy and quantitative determinate by a spectrofluorometer (Shimadzu RF-5301PC, Shimadzu, Kyoto, Japan) at an excitation wavelength of 488 nm and an emission wavelength of 529 nm. Three fields of view were randomly chosen for each treatment, and the experiment was repeated three times. The MDA content was determined as the method described previously (Wang et al., 2013) with some modifications. The mycelia were suspended in 20 mL solution containing different concentrations of UA (0, 200 and 1000 μg/mL) and incubated on a water-bath rotary shaker at 28 °C for 24 h. Sample of 1.0 g treated mycelia were homogenized with 5 mL of pre-cooled sodium phosphate buffer (100 mM, pH 7.8) mix with quartz sand. After centrifuged for 20 min (12,000 × g, 4 °C), the supernatants were collected for MDA determination. The content of MDA was tested by the thiobarbituric acid reactive substances assay, the absorbance was measured at the wavelength of 532 nm and 600 nm. The concentration was expressed as μmol/g fresh weight mycelial. Total lipid contents of A. alternata were determined with the phosphovanillin method described by (Xu et al., 2018) with some modifications. The culture and treatment of the mycelia were with the same method described in Section 2.7, then the treated mycelia were vacuum freeze-dried for 8 h. Following that a known weight of dry mycelia was homogenized with 4.0 mL of extract solution containing methanol: chloroform: water mixture (2:1:0.8, v/v/v), the mixture was dramatically shaking for extracting lipids. The samples were centrifuged at 4000 × g for 10 min, and the lower phase was taken to mix with 0.2 mL of saturated sodium chloride solution. The lipid extraction was mixed with 0.2 mL of chloroform, transferred to a new tube and 0.5 mL of H2SO4 was added, heated in a boiling water bath for 10 min and left to cool at room temperature. An aliquot of 5.0 mL of phosphoric acid-vanillin reagent was added to each tube and incubated for 15 min at 37 °C. The absorbance at 520 nm was detected. The details for the preparation of the phosphoric acid-vanillin reagent are the same as
2.9. Determination of UA treatment on quality parameters of apple fruit and defense-related enzyme activities The quality parameters of infected apple fruit were measured referred to the previous study (Ge et al., 2019b). The pulp firmness was measured using a force gauge with a 0.1–2 cm diameter flat probe (Zhejiang Tuopu Instrument Co., Ltd.). Soluble solids contents (SSC) were measured with a digital refractometer (PAL-1, Atago, Tokyo, Japan). Titratable acidity (TA) was determined by acid-base titration method. The result was expressed as the percentage of malic acid. Activities of PAL and PPO were measured by the spectroscopy methods (Ge et al., 2019b; Jiao et al., 2018). The enzyme activities were calculated as the change in U/g, taking one unit of enzyme activity as equal to 0.01 ΔOD per min. 2.10. Statistical analysis All the experiments were completed by a randomized design and done in triplicate; each replicate included three induvial determinations. Data were performed with SPSS software (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) and Duncan's multi-range test were performed to determine significant differences between the means, where the data were considered statistically significantly when p < 0.05. 3. Results 3.1. In vitro antifungal activity of UA against A. alternata UA significantly (p < 0.05) inhibited the extension of colonies and the inhibition exerted a dose-dependent manner (Fig. 1A). As the increasing concentration of UA, the colony diameters decreased significantly. When the concentration reached 200 μg/mL, the mycelial growth was inhibited by 45.6%, whereas 1000 μg/mL UA treatment roughly inhibited the mycelial growth, reached an inhibition by 70.1%. 3
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A logarithm model was used to describe the relationship between UA concentrations and inhibition rates after 9 days of incubation. The halfinhibition concentration of UA was calculated to 284.5 μg/mL, the R2 is 0.9914, indicating a good logarithmic relationship between concentrations and inhibition rates (Fig. 1A). The presence of UA inhibited spore germinations as well (Fig. 1B). The result showed that spore germination was significantly retarded (p < 0.05) exposed to UA. The spore germination rate is 82.7% in the control, whereas it was significantly inhibited by 200 μg/mL UA. The highest UA concentration (1000 μg/mL) prompted the most profound reduction of spore germination, the inhibition of 67.9% was detected. These results indicating that UA exhibited significant antifungal efficiency against A. alternata in vitro. 3.2. Effects of UA treatment on morphology changes of A. alternata Morphology alternations of spores and mycelial were observed by SEM micrographs. The spores in the control showed normal morphology of mature spores with obclavate to obpyriform structure with longitudinal and transverse septa and germ tube and warts dispersed on the surface (Fig. 2A), whereas the surface morphology of treated spores was irreversible altered after incubation exposed to 200 μg/mL UA (Fig. 2B–C). The treated spores showed significantly shriveled and recessed. Greater concavity and major broke on the surface could be observed exposed to 1000 μg/mL UA (Fig. 2C). Mycelia in the control showed the typical morphology with uniform, sturdy, thick and linearly shape (Fig. 2D), while hyphae emerged distorted and crumbled during incubation exposed to 200 mg/L UA (Fig. 2E). Seriously collapsed squashed and loss of linearity occurred in the mycelia incubated with 1000 μg/mL UA (Fig. 2F). Fig. 1. Effect of ursolic acid (UA) concentrations on in vitro growth of A. alternata. Effects of UA on colony diameter and its inhibition rate after incubation at 28 °C for 9 days (A). Effects of UA on spore germination after incubation at 28 °C for 12 h (B). The logarithm model was applied to describe the relationship between UA concentrations and inhibition rates. Untreated spores served as the control. Each value is the mean of three replicates.
3.3. Effects of UA on plasma membrane integrity, permeability and cellular leakage of A. alternata The integrity of the plasma membrane was detected qualitatively by laser confocal microscopy by PI straining. Both spores and mycelium of A. alternata grown exposed to UA showed strong red fluorescence, and the fluorescence intensity showed a dose effect (Fig. 3A). Whereas no
Fig. 2. Scanning electron microscopy images of spores (A–C) and mycelia (D–F) of A. alternata exposed to different concentrations of UA treatment. Normal morphology of spores was with the obclavate to obpyriform structure with longitudinal and transverse septa and germ tube and warts dispersed on the surface. Normal mycelia were with uniform, sturdy, thick and linearly shape. Lesioned cells showed distortion, sunken and shriveled morphology. Untreated spores and mycelia were served as the control. 4
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Fig. 3. Effects of ursolic acid on membrane integrity of A. alternata. Confocal laser scanning microscopy images showing the loss of cell plasma membrane integrity of A. alternata exposed to UA at different concentrations (A), cells with damaged plasma membranes exhibit red fluorescence. Effects of different concentrations of UA treatment on cytoplasmic leakage. Extracellular conductivity (B), proteins (C), sugars (D), nucleic acids (E) of A. alternata treated with UA. Untreated spores and mycelia were served as the control. Each value is the mean of three replicates. The vertical bars represent the standard deviations of the means.
obvious fluorescence was observed in the control demonstrating high cell membrane integrity. These results showed that UA may cause damages and alterations to cell membrane integrity thus increases the permeability and induces the debilitation or even death of the fungi. Electric conductivity value represented the permeability of A. alternata cell membranes. There was an immediate dramatic increase in extracellular conductivity detected when A. alternata were exposed to
UA at various concentrations, as well as with prolonged exposure time (Fig. 3B). With the concentration of UA was 200 μg/mL, conductivity values increased significantly (p < 0.05) after 30 min compared to the control (79.3 ± 0.98 μS/cm). The extracellular conductivity of A. alternata in the control was slightly increased, whereas it was increased steadily during the incubation when exposed to UA. At the end of incubation, the conductivity value was increased by 17.75% (p < 0.05) 5
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Fig. 4. Effects of ursolic acid on induced oxidative stress of A. alternata. Confocal laser scanning microscopy images showing the accumulation of cytosolic reactive oxygen species (ROS) of A. alternata exposed to UA at different concentrations (A), spores and mycelial with intracellular ROS induction exhibit green fluorescence. Effects of different concentrations of UA treatment on endogenous ROS production (B), lipid peroxidation (C) and total membrane lipid content (D) of A. alternata. Untreated spores and mycelia were served as the control. Each value is the mean for three replicates. The vertical bar indicates the standard deviation. Different letters indicate significant differences among sample groups (ANOVA, P < 0.05).
and 32.6% (p < 0.05) by the exposure to UA at 200 and 1000 μg/mL, respectively. The leakage of cytoplasmic contents was further determined to investigate the alteration in membrane integrity of A. alternata. The results indicated that UA induced significant leakages of soluble protein, soluble sugar and nucleic acids from A. alternata (Fig. 3C–E). The leakage of cytoplasmic contents increased rapidly after 1 h of incubation and remained increase gradually during the subsequent incubation, and the degree of leakage were positively correlated with UA concentration. These results indicated that UA induced significant damages and alterations to the cell membrane, causing leakages of cytoplasmic
content of A. alternata.
3.4. UA induced intracellular ROS production and lipid peroxidation The distribution of intracellular ROS in spores and mycelial was observed by confocal laser scanning microscopy (CLSM). As showed in Fig. 4A, both spores and mycelia cells of A. alternata grown exposed to UA showed visible green fluorescence, indicating that the accumulation of intracellular ROS, while no noticeable fluorescence was observed in the cells grown in the control. An obvious increase of fluorescence intensity observed in the cells incubated with 200 μg/mL UA. More 6
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exposure to 200 and 1000 μg/mL UA compared to the control (Fig. 4B). Antioxidant L-cysteine was used to further verification the oxidative stress effect of conidia induced by UA. As shown in Fig. 4B, the fluorescence signals were significantly quenched (p < 0.05) after the addition of L-cysteine, and maintain in a relatively low level, indicating that the increasements of fluorescence induced by UA were the result of endogenous ROS formation and accumulation. MDA can be determined as the indicator of oxidative damage of the fungal plasma membrane and could further confirm the results of the ROS assay. Compared to the control, the MDA content of the fungi exposed to 200 and 1000 μg/mL UA treatment was increased by 85.63% and 1.16-times, which demonstrating that UA treatment induced severe lipid peroxidation and caused severe lipid peroxidation in A. alternata. On the other hand, the total lipids content in A. alternata were significantly reduced (p < 0.05) by incubation exposed to UA treatments (Fig. 4D), the total lipids content in control is 19.75% and 44.52% higher compared to 200 and 1000 μg/mL UA treatment respectively, which consistent with the trend of ROS and MDA content. 3.5. Efficacy of UA in controlling black spot rot in apple fruit The development of black spot lesions in apple fruit was significantly (p < 0.05) suppressed by UA treatments (Fig. 5A). After incubation for 3 days, UA treatments significantly (p < 0.05) suppressed the startup of the disease, and on the 12th day after inoculation, lesion diameter of UA treatment group decreased by 18.76% and 34.47% respectively when compared to control. This demonstrated that UA exerts direct inhibitory effect on A. alternata, thus would inhibit the occurrence and suppress the development of black spot rot on apple fruits. In addition, UA dipping treatment could also inhibit the development of lesion, the lesion diameter was 14.95% smaller than control fruit for 12 days after inoculation. These results suggested that apart from the direct antifungal activity, UA could induce the defense responses in apple fruit against postharvest black spot rot caused by A. alternata. Notably, no surface injuries or phytotoxicity was observed in the fruit even treated with the highest concentration of UA. Also, there were no significant differences in postharvest quality parameters, including firmness, SSC, pH and TA of apple fruit among all treatments during 12 days of storage (Table 1). PAL and POD are important defense enzymes. The PAL activities in apple fruits dipping with UA were 54.51% and 11.92% higher (p < 0.05) than that in control respectively, after 3 and 6 days of storage (Fig. 5B). The activity of POD increased rapidly in fruits treated with UA and was significantly (p < 0.05) higher than that of control during storage after 3 days and the subsequent, than that of control (Fig. 5C). This indicated that UA treatment significantly activated PAL and POD enzyme activities during storage, which play an important role in induced resistance of fruit. 4. Discussion Fig. 5. Effect of UA treatments on lesion diameter of black spot rot on apple fruit inoculated with A. alternata. Effect of UA treatment (A) on lesion diameter development of apple fruit. Effect of UA immersion treatment on PAL (B) and PPO (C) activities of apple fruit. Fruits treated with distilled water were served as the control. Each value is the mean of three replicates. The vertical bars represent the standard deviations of the means. Different letters indicate significant differences among sample groups (ANOVA, P < 0.05).
Apple fruit is one of the most important fruit crops worldwide, however, postharvest decay by pathogen fungi lead to tremendous economic losses. The present work investigated the antifungal efficiency of ursolic acid, a natural triterpenoid, against A. alternata and its possible mechanism involved as well as the potential of UA to preserve apple fruit was further evaluated. We found that the colony growth and spore germination of A. alternata, as well as black spot lesions in apple fruits, could be significantly inhibited by UA treatment. The physiological function of the triterpenoid compound is to defend against plant pathogens invasion (Domingo et al., 2009). UA is the main antifungal component isolated from some medical herbs such as Rosmarinus officinalis (Collins and Charles, 1987), Breonadia salicina (Mahlo and Eloff, 2014) and Swertia chirata (Kaur et al., 2019). Previous studies have also reported the antifungal activity of UA against several postharvest fungal
intense and steady green fluorescence was observed in the cells incubated with 1000 μg/mL UA. This delegated that induced intracellular ROS generation and accumulation in the spores and mycelium of A. alternata with UA exposure. The intracellular ROS produced in the spores was determined quantitatively by fluorescence spectrophotometer. The fluorescence strength in spores was increased by 86.3% and 2.38-fold by the 7
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Table 1 Effects of UA treatment on quality parameters of apple fruit during storage at 25 °C for 12 days. Treatment
Firmness (kg/cm2)
SSC (%)
Initial UA 200 μg/mL injection UA 1000 μg/mL injection UA 200 μg/mL dipping
13.21 11.38 11.37 12.12
14.76 14.87 14.62 14.23
± ± ± ±
0.37a 0.42b 0.45b 0.32ab
± ± ± ±
pH 0.33a 0.26a 0.34a 0.35a
3.25 3.68 3.65 3.57
TA (%) ± ± ± ±
0.14a 0.12b 0.13b 0.13b
0.398 0.393 0.384 0.386
± ± ± ±
0.04a 0.04a 0.03b 0.02b
Apple fruit were divided into two groups, (I) fruits were injected with 10 μL of UA solutions at 200 and 1000 μg/mL at the wounds (5 mm deep, 2 mm wide) at equator positions of the fruit, distilled water set as control; (II) fruits were immersed with UA at 0 and 200 μg/mL for 10 min at room temperature. Firmness, soluble solids contents, pH values, and titratable acidity of apple fruit were measured on the 12th day of storage at 25 ℃. Each value is represented as mean of three replicates ± standard deviation. Values followed by different letters are significant different according to Duncan’s multiple range test (P < 0.05).
postharvest Alternaria rot on fruits. Further, its effects on the defense system of fruits were investigated. Our results showed that UA immersion treatment would suppress the development of black spot rot on apple fruit and the activation activity of PAL and POD were detected. PAL is a key enzyme in the metabolism of plant phenylpropanoid, which is essential for the biosynthesis of secondary metabolites and plays an important role in the normal growth and development of plants and defense against pathogens (Jiao et al., 2018). POD is associated with the strengthening of plant cell walls by catalyzing lignin biosynthesis, which act as an effective barrier against pathogen penetration and spread (Kärkönen and Koutaniemi, 2010). The enhanced PAL and POD activity is associated with the increase of resistance system, such as in apple (Ge et al., 2019a), peach (Jiao et al., 2018) and pear (Yu et al., 2014) fruit. A study by Shaik et al. (2016) also reported the exogenous ursolic acid spray treatment would inhibit sorghum grain mold by inducing salicylic acid production, which is associated with the enhanced plant resistance system. These findings suggest that UA could induced the defense responses in apple fruit against the postharvest A. alternata invasion. The existence of reports on the synthesis of UA reveals the scope of its commercialization (López-Hortas et al., 2018) encouraging us to study the wide range of application in the future. Additionally, further studies should be made to dissect the antifungal membrane binding sites of UA in more detail and its molecular mechanisms underlying in more details.
species (Jabeen et al., 2011; Mahlo and Eloff, 2014; Shaik et al., 2016). Our results suggest that UA exerts in vitro antifungal activity and reduce disease development in apple fruit. The plasma membrane is an important target for antifungals. Considering the lipophilic character of UA, we investigated whether UA interacts with the cell membrane of A. alternata. The morphology observation indicated the prominent damage on spores and mycelial cell wall after UA treatment, which suggested that UA may induced membrane disturbance resulted in disruption of the cell wall and lysis of the cell. UA may preferentially partition from the aqueous phase and penetrate and accumulate in the phospholipid bilayer, which would damage the phospholipids layers and induces the decreased vitality of the cell (da Cruz Cabral et al., 2013). The spores and mycelia treated by UA could be stained with PI and the fluorescence intensity showed a dose effect, which indicated the lesioned membrane integrity. Our result was in line with Xu et al (2018) who found an increased influx of PI into A. alternata exposed to cinnamaldehyde, a hydrophobic monoterpene compound. Meanwhile, was detected a significant leakage of cytoplasmic content of A. alternata treated with UA in this study. The lipophilic property and hydrophobic scaffold of UA could bind to the hydrophobic region of proteins and dissolve in lipid phases, leading the impairment of membrane-bound proteins which maintain homeostasis in the plasma membrane (Si et al., 2018). The cytoplasmic content of A. alternata may leak into the extracellular fluid due to the damage of cell membrane during growth. Ji et al. (2018) reported that methyl thujate treatment increased the intracellular macromolecular cytoplasmic components leakage, which is associated with the damaged cell membrane integrity. These results suggest that UA exerts antifungal activity via the membrane-targeted mechanism. Disruption of membrane function and integrity due to ROS oxidation of membrane lipids is part of some of the antifungal potent modes of action (Rautenbach et al., 2016). The current study indicated that the fluorescence intensity of ROS was enhanced obviously in the treated spores and mycelia, and it was positively correlated with the concentration of UA. In addition, the enhanced fluorescence intensity was quenched in the presence of the oxygen scavenger L-cysteine, which indicated the increase of fluorescence was indeed because of the ROS formation that contributes to contributes to its antifungal efficiency (Helmerhorst et al., 2001). Our results were consisting with the previous study which reported that UA treatment and plant extracts enriched in UA could induce ROS production in microorganisms (Oloyede et al., 2017). Redundant ROS would cause lipid peroxidation by attacking unsaturated fatty acids distributed in lipids. MDA is the hallmark product of lipid peroxidation, the MDA content increased significantly in UA treated group, suggesting UA induced serious lipid peroxidation and massive oxidative damage to the membrane occurred in A. alternata. Correspondingly, the result was supported by the decreased content of membrane lipids. Our results suggest that UA exerts directly antifungal activity through membrane damage and oxidative stress against A. alternata which ultimately lead to the collapse and death of the cells. The directly antifungal activity of UA on A. alternata revealed that it could be potentially used as an alternative fungicide for controlling
5. Conclusion In summary, ursolic acid could effectively inhibit A. alternata growth by the mode of membrane-targeted mechanism. UA induced the disturbance of membrane permeability, disruption of membrane integrity, and intracellular ROS accumulation in A. alternata result in the lysis of the pathogen, thus exert directly antifungal activity. In addition to its direct antifungal activity, UA induced the defense responses in apple fruit against postharvest black spot rot caused by A. alternata. UA is a triterpenoid widely distributed in many plant species, which suggest that application of UA may provide a novel effective strategy to control postharvest diseases of fruits. Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work. Acknowledgement This research was supported by the national key research and development projects of the China Ministry of Science and Technology (No. 2018YFD0401302). References Cargnin, S.T., Gnoatto, S.B., 2017. Ursolic acid from apple pomace and traditional plants:
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