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6-Benzylaminopurine inhibits growth of Monilinia fructicola and induces defense-related mechanism in peach fruit
Food Chemistry 187 (2015) 210–217
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6-Benzylaminopurine inhibits growth of Monilinia fructicola and induces defense-related mechanism in peach fruit Yangyang Zhang, Lizhen Zeng, Jiali Yang, Xiaodong Zheng, Ting Yu ⇑ College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food Technology and Equipment, Zhejiang University, Hangzhou 310058, People’s Republic of China
a r t i c l e
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Article history: Received 2 December 2014 Received in revised form 5 April 2015 Accepted 21 April 2015 Available online 23 April 2015 Chemical compounds studied in this article: 6-Benzylaminopurine (PubChem CID: 62389) Superoxide dismutase (PubChem CID: 5359597) Malondialdehyde (PubChem CID: 10964)
a b s t r a c t This study demonstrated the inhibitory effect of 6-benzylaminopurine (BAP), the first generation synthetic cytokinin, on the invasion of Monilinia fructicola in peach fruit and the possible mechanism involved for the first time. Our results suggested that BAP treatment had a 63% lower disease incidence and approximately 10 times lower lesion diameter compared to the control throughout the incubation period. In vitro BAP showed a direct inhibitory effect on M. fructicola spore germination. BAP could prevent fruit texture deterioration and protect the cell membrane from oxidative stress, while no adverse effects were observed on fruit quality maintenance. Analysis of defense-related enzymes activities indicated that the use of BAP induced higher specific polyphenol oxidase and peroxidase activities which triggered stronger host defensive responses. Thus, our results verified the proposed mechanism of BAP in controlling M. fructicola by direct inhibitory effect, delay peach senescence and activation of defensive enzymes. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: 6-Benzylaminopurine (BAP) Monilinia fructicola Peach Postharvest
1. Introduction Peaches are one of the most important fruit crops cultivated in east and north areas of China with an estimated annual production approximate 12 million metric tons in 2012 (Food, 2012). Brown rot, caused by Monilinia fructicola, is a serious bacterial disease that affects peaches quality from blossom period to harvest storage, with broad devastating dissemination causing production loss estimated at 20% or more (Zhu, Chen, Luo, & Guo, 2005). Due to the potential hazard of synthetic fungicides on human being’s health and the environment, none of the fungicide spray programs are authorized for postharvest control of M. fructicola. Hence there is a growing interest in alternative strategies for M. fructicola inhibition (Casals et al., 2012). Within these alternatives there are three main fields (physical control, biological agents and chemicals). The use of cold storage, radio frequency and hot water treatment has been proposed to ⇑ Corresponding author at: 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China. E-mail address: [email protected] (T. Yu). http://dx.doi.org/10.1016/j.foodchem.2015.04.100 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
control brown rot on peaches (Spadoni, Neri, Bertolini, & Mari, 2013). Some biological agents have shown satisfactory efficacy for M. fructicola control, such as Bacillus subtilis CPA-8 and Aureobasidium pullulans strains (Mari, Martini, Guidarelli, & Neri, 2012). Nevertheless, these methods have some drawbacks and limitations in commercial fruit production. Physical treatments need large equipment to process fruit and may damage sensory quality, while biocontrol agents are limited by the low effectiveness. (Rungjindamai, Jeffries, & Xu, 2014; Sholberg & Kappel, 2008). Applications of chemicals with non-toxicity provide effective inhibition against postharvest decay and high value of commercial promotion as well. Several natural chemical compounds, such as chitosan, tea polyphenol and carnauba wax (Gonçalves, Martins, Junior, Lourenço, & Amorim, 2010; Ma, Yang, Yan, Kennedy, & Meng, 2013), have demonstrated direct inhibitory actions on M. fructicola growth. Therefore, non-toxic chemicals seem to be the promising alternatives to the synthetic fungicides. Cytokinins are involved in various aspects of plant biological processes, ranging from organ formation and apical dominance to leaf senescence (Hwang, Sheen, & Müller, 2012). 6Benzylaminopurine (BAP), the first generation synthetic cytokinin,
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plays a significant role in plant cell division and enhancing chilling tolerance (Chen & Yang, 2013). Moreover, BAP has been approved as biopesticide in postharvest practices by the United Stated Environmental Protection Agency (US-EPA). In harvest commodities, spray application of BAP is involved in extending the vase life of pot tulips and improving quality maintenance of summer squash (Kim & Miller, 2009). Direct evidences concerning the applications of cytokinins on postharvest pathogens control are scarce, little information evaluates the efficiency of BAP alone on brown rot control in peach fruit. The objectives of this study were to determine the efficiency of different concentrations of BAP in controlling peach brown rot caused by M. fructicola during the postharvest storage and the possible mechanisms involved.
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2.4. Effect of time between BAP treatment and pathogen inoculation on inhibition of M. fructicola To assay the effect of time between BAP treatment and pathogen inoculation on inhibition of M. fructicola, peach fruit were washed, sterilized and wounded following the method described above. Fifty microliters of 500 mg/l BAP was added into the wounds of peach fruit. After a series time (0, 6, 12 and 24 h) of BAP treatment, thirty microliters of M. fructicola spore suspension (1 104 spores ml1) was inoculated into each wound as well. Fruit without BAP treatment acted as the inoculated control. Then fruit were stored in the thermostatic cultivation room at 25 °C for recording the disease incidence and lesion diameter. All treatments were conducted with three replicates and ten fruit per replicate, which were repeated twice.
2. Materials and methods
2.5. Effect of BAP on spore germination in PDB
2.1. Fruit and chemicals
The effect of BAP on spore germination of M. fructicola was assayed in PDB. Pure colonies of M. fructicola were used to prepare suspensions concentration of 1 105 spores ml1. One fifth of a milliliter of each suspension blended with 1 ml BAP at final concentrations of 1, 10, 100, 200, 500 and 1000 mg/l were distributed into a test tube containing 0.8 ml PDB, sterile distilled water was used as control. All test tubes were incubated at 25 °C for 24 h in the dark. Approximately 100 spores of M. fructicola were measured randomly in the microscopic visual field for germination rate and germ tube length. Each treatment included three test tubes randomly within three replicates.
Peach (Prunus persica L. Batsch. cv. Hanlumi) were harvested at the commercial mature stage in Shandong Province, China and sorted based on the size and the absence of physical injuries or disease infection. Prior to treatments, fruit were sterilized in a water solution of 0.1% sodium hypochlorite for 2 min, rinsed with tap water completely and then desiccated at room temperature. An isolate of M. fructicola was obtained from decayed peach fruit and cultured on potato dextrose agar (PDA) medium at 25 °C in the dark. Sporangiospore suspension was prepared from the flooded 7-days-old cultures and suspended in sterile distilled water. Afterwards the spore concentration was observed by the means of a hemocytometer and adjusted to 1 104 spores ml1 with sterile distilled water. BAP was purchased from Sinopharm Chemical Reagent Company (Beijing, China). The stock solution of BAP was diluted to a series (1, 10, 100, 200, 500 and 1000 mg/l). 2.2. Effect of BAP on inhibition of M. fructicola in vivo Each peach fruit was wounded softly with the sterile punch in the equatorial region to form a hole (5 mm deep and 5 mm in diameter). Fifty microliter of BAP at different concentration was added to the wound separately in each treatment: BAP at different concentrations (100, 200, 500, 1000 mg/l) and sterile distilled water served as control. Thirty microliters of M. fructicola spore suspensions at 1 104 spores ml1 were inoculated into the wounds of all peach fruit 1 h later. Fruit were placed into plastic trays and packaged with preservative film in the thermostatic cultivation room for incubation at 25 °C with high relative humidity (90–95%). The number of infected peaches was monitored daily. Three replicates of 20 peaches were selected as a unit for each treatment, the disease incidence and lesion diameter of total peaches were recorded for statistical analysis, respectively. 2.3. Effect of BAP on inhibition of M. fructicola at low temperature Peach fruit were wounded in each fruit equatorial region as above. Each wound was treated with fifty microliter of BAP in a series of concentration: 100, 500 and 1000 mg/l; wounds treated with equal volumes of sterile distilled water served as controls. Twenty peach fruits were packaged as a group and thirty microliters of M. fructicola (1 104 spores mL1) spore suspensions were pipetted into the wounds. Afterwards all fruit were incubated at 4 °C in refrigerated cabinet. All treatments were conducted with three groups as replicate.
2.6. Effect of BAP on survival of M. fructicola on PDA M. fructicola spore suspension was mixed with serial concentrations of BAP (0, 1, 10, 100, 200, 500 and 1000 mg/l) in order to adjust the final concentration to 100 spores ml1. The mixed suspensions were poured onto petri dishes (60 mm in diameter) with 15 ml PDA, respectively. Finally colonies per plate were calculated after 72 h incubation at 25 °C. Three petri dishes were chosen randomly as a replicate and each treatment included three replicates. 2.7. Effect of BAP on defense-relate enzymes activities in vivo For the enzyme assay, fifty microliters of 500 mg/l BAP was added to each wound and the same dose of sterile distilled water was regarded as control. The fruit tissues near the wounds were sampled after a series of time interval (0, 6, 12, 24, 36 and 48 h) and frozen in the liquid nitrogen immediately. Each sample treatment made up of three fruit tissues was prepared for the assay of enzyme activities. Each frozen tissue sample (0.2 g) was homogenized with 1 ml of sodium phosphate buffer (50 mM, pH 7.8) comprising 1.33 mM Ethylenediaminetetraacetic acid and 1% (w/v) polyvinyl-pyrrolidone (PVP) and ground at 4 °C. The homogenate was centrifuged at 12,000g for 10 min, then the extracts obtained were used for the enzyme assay of superoxide dismutase (SOD), polyphenol oxidase (PPO) and peroxidase (POD) at 4 °C. SOD activity was determined by the method of using colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), following the manufacturers specifications. Results of SOD activity was expressed as units per lg of protein. PPO activity was measured by using pyrocatechol as a substrate following the method of Aquino-Bolaños and Mercado-Silva (2004). Fifty microliters crude enzyme was blended with 250 ll 50 mM sodium phosphate buffer (pH 6.4) containing 10 mM pyrocatechol, the reaction was incubated for 1 min at 30 °C, then the absorbance of the supernatant was recorded once 30 s for 10 times
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at 398 nm using a spectra max plus 384 (Molecular Devices Limited, USA). One unit of PPO activity was defined as the amount of enzyme extracts producing an increase of A398 nm by 0.01 in 1 min and the activity was expressed as units per lg of protein. POD activity determination was operated according to the method of Lurie, Fallik, Handros, and Shapira (1997). The reaction mixture was made up of 220 ll of 0.3% (v/v) guaiacol and 20 ll of crude enzyme extracts. The reaction was started by the addition of 60 ll of 0.3% (w/v) H2O2, later the change of absorbance at 460 nm was measured once 30 s during the whole process of 5 min. One unit of POD activity is defined as the amount of enzyme required to cause an increase in absorbance of 0.01 at 470 nm per minute, the enzyme activity was expressed as units per lg of protein. MDA content was measured according to the method of Du and Bramlage (1992), with some modification. A fifth of one gram of frozen tissue was ground with 1 ml 10% (w/v) trichloroacetic acid buffer and the homogenate was centrifuged at 12,000g for 10 min at 4 °C. Enzyme preparation containing 700 ll of crude enzyme and the same volume of 0.6%(w/v) thiobarbituric acid was incubated for 10 min at 100 °C. After that the reaction mixture was cooled in ice immediately and centrifuged at 4000g for 15 min at 4 °C, the volume of supernatant was measured also. The absorbance of reaction mixture was measured at 532, 600 and 450 nm, respectively, while 0.6% (w/v) TBA was regarded as blank. MDA was calculated according to the formula:
C MDA ðlmol=lg proteinÞ ¼ ð6:45 ðA532 A600 Þ 0:56 A450 Þ ðextract volumeÞ=ðprotein weightÞ Protein content of each enzyme assay was determined according to Bradford (1976) with bovine serum albumin (Sigma Chemicals Co., St. Louis, USA) as standard. Each treatment contained three replicates and the experiment was repeated twice.
collected from opposite regions at the equator of fruit were shaped into discs of 8 mm thick and 2 g weight. Then samples were immersed in 5 ml of deionized water for 10 min to fill the fruit cells with deionized water, subsequently samples were placed in the vacuum desiccator at 0.06 MPa for 10 min. The reaction mixture was incubated on a rotary shaker at 150g for 1 h at room temperature. After briefly vortexing, electrolyte conductivity was determined with a conductivity meter (Model DDS-11A, Shanghai Scientific Instruments) as initial (c1), afterwards samples were treated with boiling water heating for 15 min and the electrolyte conductivity were second recorded (c0) after cooled down to room temperature. The electrolyte conductivity was defined as follows:
Electrolyte conductivity ¼ c1 =c0 100% To evaluate the content of phenolic, flavonoids and anthocyanin, fruit samples were prepared as described above for determinations. The supernatant was extracted from 2 g of frozen tissue with 20 ml ice-cold 1% HCl–methanol solution and then centrifuged at 15,000g for 15 min at 4 °C. 1% HCl–methanol solution was regarded as blank, accordingly phenolic content was defined as OD280/g, flavonoids content was expressed as OD325/g and anthocyanin content equaled to (OD530 OD600)/g.
2.9. Data analysis All statistical analyses were performed with SPSS 19.0 statistical software by one-way analysis of variance and Duncan’s multiple range tests. When the number of means in each group is two, the independent samples Tukey’s test is applied for means separation. p-Values of different treatments less than 0.05 were considered to be significant.
2.8. Effect of BAP on peach quality
3. Results
The effect of BAP on fruit quality was investigated on peach fruit. Fruit were immersed in solutions of sterile distilled water (control) and 500 mg/l BAP, respectively for 10 min and then incubated in cold storage cabinet at 4 °C. Fruit quality parameters in each treatment during storage was measured by monitoring changes of the fruit at 16 days. At the sampling date, peach from both control and BAP-treated groups were taken for quality evaluation (firmness, titratable acidity and membrane permeability). The determination of fruit firmness was conducted according to the method of Cao, Jiang, and Zhao (2011, chap. 2). The samples were peeled to 1 mm, fruit firmness was made at two equidistant points on the equator of each fruit with a hand-held penetrometer (GY-2, Fruit Firmness Tester, Zhejiang, China). The average of those two results was considered as one replicate. Ten fruit per treatment were determined and data are expressed in megapascal (MPa). Titratable acidity (TA) content was assayed by means of Cao et al. (2011, chap. 2). Samples of the mashed fruit (5.0 g) were centrifuged by 4000g for 10 min and the supernatants were used for titrating to pH 8.1 with 0.01 mol l1 NaOH, phenolphthalein functioned as the indicator. TA content was calculated as percentage of malic acid which is multiplied by the conversion factor of organic acid concentration per gram of fresh mass. Each treatment was consisted of three replicates and per replicate was pooled from three wounds that were collected from three fruit, the experiment was performed twice. For the membrane permeability assay, membrane permeability was evaluated on the basis of electrolyte leakage according to the method of Lutts, Kinet, and Bouharmont (1996) with slight modification. The percentage loss of electrolyte was calculated. Samples
3.1. Effect of BAP on inhibition of M. fructicola in vivo Fruit deterioration was significantly reduced by BAP treatments in vivo in a concentration dependent manner (Fig. 1A and B). After 60 h incubation, the disease incidence reached values of 77.3% and 2.6% in control and 100 mg/l BAP-treated fruit. BAP at concentrations of 500 and 1000 mg/l significantly suppressed disease incidence throughout incubation period and resulted in 49.4% and 38.7% at 108 h, respectively. Average lesion diameter values of those treatments were generally 10 times lower than that of control. When the concentration of BAP treatment increased from 500 to 1000 mg/l, no significant differences were observed in the disease incidence and average lesion diameter.
3.2. Effect of BAP on inhibition of M. fructicola at low temperature Fig. 1C showed the brown rot incidence of different concentrations of BAP at 4 °C storage. The wounds inoculated with BAP were effective in inhibiting the M. fructicola infection. BAP at lower-dose treatment was not sufficient to control rot occurrence at 228 h. While no appearance of brown rot was detected in 500 mg/l BAP treatment until 336 h and the peak of disease incidence reached 33.3% at the end period of storage. Average lesion diameters were significantly lower than those in control treatment (Fig. 1D). Disease incidence of 1000 mg/l BAP treatment resembled that of 500 mg/l BAP treatment but peaked at 46.7%. Low temperature storage could extend the incubation time approximately 4-fold than that in 25 °C storage, whereas it could provide enduring protection of peach fruit from the pathogen attack.
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Fig. 1. Inhibitory effect of 6-benzylaminopurine (BAP) at different concentrations on growth of M. fructicola in peach fruit. Disease incidence (A) and average lesion diameter (B) of peach fruit infected by M. fructicola were investigated for 108 h at 25 °C. Serial concentrations of BAP at 100, 200, 500 and 1000 mg/l were pipetted into fruit wounds to suppress the growth of M. fructicola (1 104 spores ml1) in vivo, using sterile distilled water as a control. Disease incidence (C) and average lesion diameter (D) of peach fruit infected by M. fructicola were recorded throughout incubation period at 4 °C. Fruit wounds were treated with sterile distilled water (as control) and BAP at various concentrations: 100, 500 and 1000 mg/l to inhibit the growth of M. fructicola (1 104 spores ml1). Vertical bars represent standard errors of three replicates.
3.3. Effect of treatment time interval on inhibition of M. fructicola Results above had indicated that peach fruit treated with 500 mg/l BAP showed a remarkable suppression on M. fructicola growth (Fig. 1). The percentage of infected fruit decreased when the treatment time interval between BAP treatment and inoculation of M. fructicola was 12 h or longer (Fig. 2A). After 24 h incubation, disease incidence reached the values of 18.7% and 25.3% in 12 and 24 h treatment, respectively, which were generally lower than
that in 0 h treatment at the value of 78.8%. There was no significant difference in disease incidence and average lesion diameter between 12 and 24 h treatment time interval. 3.4. Effect of BAP on spore germination of M. fructicola in PDB BAP treatments suppressed the spore germination of M. fructicola in a dose-dependent manner (Fig. 3A). Percentage of spore germination was 93.3% and 81.2% for control and 10 mg/l BAP
Fig. 2. Inhibitory effect of time interval between 6-benzylaminopurine (BAP) treatment and inoculation of M. fructicola in peach fruit at 25 °C. Thirty microliters of M. fructicola suspension (1 104 spores ml1) was inoculated at a serial time interval (0, 6, 12 and 24 h) after fifty microliters of 500 mg/l BAP was added into fruit wounds. Disease incidence (A) and average lesion diameter (B) of peach fruit infected by M. fructicola were investigated after 84 h incubation. Standard errors of three replicates were described as vertical bars.
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Fig. 3. In vitro antifungal activity of 6-benzylaminopurine (BAP) against M. fructicola throughout incubation at 25 °C. Effect of BAP at different concentrations (10, 100, 200, 500 and 1000 mg/l) on spore germination of M. fructicola in peach fruit after incubation at 25 °C in PDB for 24 h (A). Effect of BAP at different concentrations (1, 10, 100, 200, 500 and 1000 mg/l) on survival of M. fructicola 72 h after inoculation on PDA (B). Vertical bars represent standard deviations of the means. Significantly different values are identified with different letters according to Duncan’s multiple range test at p < 0.05.
treatment, respectively. When the concentration of BAP increased to 200 mg/l, spore germination (Fig. 3A) and germ tube elongation (not shown) of M. fructicola was significantly inhibited. Moreover, the germination rate in 1000 mg/l BAP treatment was 70.2% lower than that of control, indicating that BAP retarded the growth of M. fructicola spores in vitro. 3.5. Effect of BAP on survival of M. fructicola on PDA Fig. 3B illustrated the inhibitory effect of BAP on colony growth of M. fructicola on PDA. Suppression on M. fructicola colony depended on the concentration of BAP treatment: the higher concentration, the stronger inhibition. BAP at concentration of 100 mg/l exhibited significantly colony reduction (p < 0.05) compared with that in control. When the application of BAP concentration increased to 1000 mg/l, the value of average colony number was merely 3.7 colonies per plate. In all BAP treatments, values of average colony number were generally lower than the control at 56.9, which demonstrated that BAP was assuredly lethal to spore germination of M. fructicola in vitro. 3.6. Effect of BAP on defense-related enzymes activities Fig. 4 indicated the change pattern of defense-related enzymes relying response to BAP. The activity of SOD in BAP treatment increased continuously up to 48 h, an increment in SOD activity was detected at 24 h (p < 0.05), which was approximately 4-fold higher than that in control (Fig. 4A). SOD activity in control was generally lower than that in BAP-treated samples throughout incubation period. PPO activity in BAP treatment peaked at 12 h and then declined to 48 h (Fig. 4B). In contrast, control samples showed relatively stable in PPO activity. In BAP treatments, POD activity rapidly started to increase at 6 h and reached a peak at 12 h, in correlation with the induced PPO activity in peach (Fig. 4C), while little change of POD activity was observed in the control. In all samples, the accumulation of MDA started to increase at 6 h and continuously increased during incubation (Fig. 4D), however the application of BAP decreased MDA accumulation to a lesser extent than that of control samples. 3.7. Effect of BAP on peach quality The quality parameters were measured after 16 days storage at 4 °C. Fruit firmness is the most significant characteristic reflecting sensory quality of peach fruit (Table 1). Fruit firmness decreased
significantly to 9.38 MPa in the control, which showed up a little soft rotten. Firmness of BAP-treated fruit was 13.8 MPa showing no marked change during the storage. Moreover, no significant differences of firmness were recorded in BAP-treated peach throughout storage. Level of titratable acidity decreased in both control and BAP-treated fruit during refrigerate storage, but no differences were found between both groups of peach. An increase in cell membrane permeability indicated the permeation of cytoplasm and cell membrane damage, the values of cell membrane permeability were 59.2% and 48.3% for control and 500 mg/l BAP treatment, respectively, although an increment in BAP-treated fruit was observed after 16 days, the cell membrane permeability delayed by BAP was still significantly lower than that of the control. Change of total phenolic content in the peach fruit are shown in Table 2, an increasing level of total phenolic was found at the end of storage period, but no differences were found between control and BAP-treated fruit. The same trends were also observed in flavonoids and anthocyanins accumulation, the content of flavonoids and anthocyanins showed a slight increment in all treatments but was close to that in initial treatment, there were no statistical differences between control and BAP treatment. The present results indicated that BAP treatment could prevent fruit firmness deterioration and delay cell membrane permeability damage, while no adverse effects were observed on titratable acidity and total phenolic accumulation.
4. Discussion Previous studies had reported that cytokinins integrated with other measures on fruit were effective in reducing postharvest pathogen infection by inhibitory effect against postharvest pathogen and inducing defense system in plant host (Yu, Wang, Yin, Feng, & Zheng, 2008). Results from this study indicated that BAP directly inhibited the growth of M. fructicola in vivo and it obtained satisfactory control of M. fructicola throughout the incubation when the concentration increased to 500 mg/l or above (Fig. 1A and B), this data was similar to an earlier report by Zheng, Yu, Chen, Huang, and Wu (2007), where they found the application of BAP was sufficient to suppress Penicillium expansum infection in the early stage. Moreover, cold storage could greatly prolong the incubation time 4-fold longer than that at the optimal temperature of 25 °C (Fig. 1C and D). To our knowledge, this is the first work showing that BAP can effectively suppress M. fructicola infection in peach fruit. Homoplastically, results of assays in vitro indicated the direct inhibitory effect of BAP on spore germination
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Fig. 4. Effect of 6-benzylaminopurine (BAP) on the activities of superoxide dismutase (SOD) (A), polyphenol oxidase (PPO) (B), peroxidase (POD) (C) and malondialdehyde (MDA) content (D) in peach fruit. Peach fruit treated with fifty microliters of 500 mg/l BAP and aliquot of sterile distilled water (as control) were sampled at a series of time point (0, 6, 12, 24, 36 and 48 h) after inoculation. Asterisks indicate significant differences between BAP treatment and control sample. Vertical bars represent standard deviations of the means. Significantly different values are identified with different letters according to Duncan’s multiple range test at p < 0.05.
Table 1 Effect of 6-benzylaminopurine on peach fruit quality parameters stored after 16 days at 4 °C. Treatment
Firmness (MPa)
Titratable acidity (%)
Membrane permeability (%)
Initial 16 days control 16 days BAP
13.94 ± 0.97a 9.38 ± 2.17b
0.24 ± 0.01a 0.16 ± 0.02b
34.79 ± 1.37c 59.17 ± 4.29a
13.80 ± 3.05a
0.16 ± 0.02b
48.29 ± 3.12bc
Peach fruit were immersed in sterile distilled water and the solutions of 500 mg/l BAP for 10 min and air-dried subsequently. Samples were obtained after 16 days incubation at 4 °C. Values identified with the different letter in columns show significant difference on the basis of Duncan’s multiple range test (p < 0.05), each value represents a mean of three replicates (n = 3).
Table 2 Effect of 6-benzylaminopurine on peach fruit quality parameters (total phenolic, flavonoids and anthocyanins) after 16 days incubation at 4 °C. Treatment
Total phenolic (DOD/g)
Flavonoids (DOD/g)
Anthocyanins (DOD/g)
Initial 16 days control 16 days BAP
7.84 ± 0.48a 8.50 ± 0.55a
8.18 ± 0.48a 8.99 ± 0.60a
0.015 ± 0.002a 0.018 ± 0.004a
9.33 ± 0.50a
9.79 ± 0.49a
0.015 ± 0.000a
Peach fruit were immersed in sterile distilled water and the solutions of 500 mg/l BAP for 10 min and air-dried subsequently. Samples were obtained after 16 days incubation at 4 °C. Values identified with the different letter in columns show significant difference on the basis of Duncan’s multiple range test (p < 0.05), each value represents a mean of three replicates (n = 3).
of M. fructicola in vitro. BAP has been considered as nontoxic substance for the postharvest applications by US-EPA. Therefore BAP, due to its non phytotoxicity and antimicrobial activity, supports the possibility of a promising alternative strategy for postharvest brown rot management. Furthermore, BAP treatment was also found to delay softening in peach fruit efficaciously without adverse effects on quality parameters of peaches. This observation may be associated with the tissue susceptibility to dehydration and disassembly of cell wall polysaccharides (Vicente, Saladie, Rose, & Labavitch, 2007). Consistent with this possibility, Massolo, Lemoine, Chaves, Concellón, and Vicente (2014) reported that effect of BAP on firmness maintenance might be attribute to cell wall reinforce. BAP is also an inhibitor of respiratory kinase in plants and increases postharvest life of green vegetables in the field of agriculture (Ge et al., 2011), the firmness maintenance demonstrated that delay of peach senescence, corresponding to the application of BAP, resulted in an enhanced protection against pathogen invasion, which provided a hypothesis to explain the role of BAP in plant defense system. Interestingly, our results showed that BAP was more effective against M. fructicola infection when the time interval between BAP treatment and pathogen inoculation was up to 24 h in fruit wounds. This observation suggested that more efficient inhibition of brown rot in peach may be associated with host-mediated resistance induced by BAP. In general, plants can acquire enhanced resistance to pathogen infection when response to triggering factor such as chemical inducers. Although, not much is known about the molecular mechanism underlying this process, it does require a
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time frame within which it occurs from signal generation to robust activation of host defense in the systemic tissues (Kachroo & Robin, 2013). For example, previous reports indicated that chitosan, a chemical elicitor, could directly inhibit the growth of M. fructicola in peach fruit, but also initiate defensive mechanisms against pathogenic attacks (Yang, Zhang, Bassett, & Meng, 2012). In addition, it was worth noting that when the time interval reached 24 h, disease incidence was approximately three times lower than that in 0 h treatment, which showed that the improvement of induced defense mechanism could be the major factor underlying the effect of BAP, direct inhibition on the growth of M. fructicola might be the rapid and more efficient measures. Recent findings had suggested that the mechanism of induced resistance was involved in the activation of defense enzymes, accumulation of antifungal compounds, pathogen-resistant protein, increasing of reactive oxygen species and lignification of epidermal cells. (Vilanova et al., 2014). Our results showed an increment in SOD activity was detected in BAP-treated peach, which was consistent with the results reported by Xu et al. (2011). SOD, identified as essential component in antioxidant defense, converts superoxide radicals into hydrogen peroxide and oxygen. Within a cell, SOD constitute the first line of detoxification of reactive oxygen species (ROS) to prevent the destruction of the cell membrane in pathogen invasion (Alscher, Erturk, & Heath, 2002). MDA has been recognized as a relevant product of polyunsaturated fatty acids, which reflects the oxidative damage of cell membrane under adversity environment (Del Rio, Stewart, & Pellegrini, 2005). Current study showed a decrement of MDA accumulation in peach tissues inoculated with BAP even though MDA level was not completely suppressed. This result was in accordance with a previous study by Xu et al. (2011), where they suggested BAP was useful to delay the MDA accumulation within the cell. Apart from MDA content, permeability of cell membrane could be potentially related to the cell membrane damage, it has been shown that BAP was effective in cell membrane protection (Zavaleta-Mancera et al., 2007). In the present study, this must be explained by a mechanism in which the high level of antioxidant enzymes induced by BAP and protection of cell from oxidative stress could delay plant host senescence in order to restrain infection by the pathogen (Xu et al., 2014). POD and PPO are known to play a key role in fruit host defense mechanism (Liu, Jiang, Bi, & Luo, 2005). POD activity was also associated with lignification resulting in reinforcement of cell walls warding off pathogen penetration (Hofrichter, 2002). Higher activity of POD was induced in wounds inoculated with BAP compared to the control treatment. Similarly, an increase in POD activity in apple fruit, as a result of gibberellic acid treatment, was found to enhance plant resistance to grey mould rots (Yu & Zheng, 2007). Our results indicated that an increment in PPO activity, detected after the application of BAP, might be associated with producing antimicrobial phenolic substances. A previous report by Wei and Ye (2011) supported this result, they demonstrated that activities of PPO and POD were enhanced in green asparagus treated with BAP. PPO is a ubiquitous copper-containing enzyme in plants and probably involves in catalyzing the oxygen-dependent oxidation of phenols to quinones (Mayer, 2006). Previous studies indicated that PPO was associated with the lignification of plant cells and suberin formation during the microbial invasion (Lee et al., 2012). While the PPO-overexpressing tomato plants exhibited a great increase in plant defense against Pseudomonas syringae in the infection process (Li & Steffens, 2002). These results suggested a positive correlation between the enhancement of POD and PPO activity and the stronger induced defense responses. In conclusion, results in this study demonstrated that BAP could directly inhibit the growth of M. fructicola and postpone the deterioration in postharvest peach fruit. The delay of fruit senescence and enhanced activities of defensive enzymes had improved fruit
immunity against M. fructicola. Moreover, BAP did not affect the peach edible quality throughout storage. More intensive investigations should be carried out to optimize certain formulation strategies, such as biocontrol agents and low-risk chemicals, integrated with BAP to maximize the efficacy. Acknowledgements This research was partially supported by the National Natural Science Foundation of China (31471627) and the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (201061). References Alscher, R. G., Erturk, N., & Heath, L. S. (2002). Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany, 53(372), 1331–1341. Aquino-Bolaños, E. N., & Mercado-Silva, E. (2004). Effects of polyphenol oxidase and peroxidase activity, phenolics and lignin content on the browning of cut jicama. Postharvest Biology and Technology, 33(3), 275–283. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72, 248–254. Cao, J. K., Jiang, W. B., & Zhao, Y. M. (2011). Experiment guidance of postharvest physiology and biochemistry of fruits and vegetables (1st ed.). Beijing: China Light Industry Press. Casals, C., Elmer, P. A. G., Viñas, I., Teixidó, N., Sisquella, M., & Usall, J. (2012). The combination of curing with either chitosan or Bacillus subtilis CPA-8 to control brown rot infections caused by Monilinia fructicola. Postharvest Biology and Technology, 64(1), 126–132. Chen, B., & Yang, H. (2013). 6-Benzylaminopurine alleviates chilling injury of postharvest cucumber fruit through modulating antioxidant system and energy status. Journal of the Science of Food and Agriculture, 93(8), 1915–1921. Del Rio, D., Stewart, A. J., & Pellegrini, N. (2005). A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutrition, Metabolism and Cardiovascular Diseases, 15(4), 316–328. Du, Z., & Bramlage, W. J. (1992). Modified thiobarbituric acid assay for measuring lipid oxidation in sugar-rich plant tissue extracts. Journal of Agricultural and Food Chemistry, 40(9), 1566–1570. Food and Agriculture Organization of the United Nations. (2012). URL Accessed 27.06.13. Ge, X., He, J., Yang, Y., Qi, F., Huang, Z., Lu, R., et al. (2011). Study on inclusion complexation between plant growth regulator 6-benzylaminopurine and bcyclodextrin: Preparation, characterization and molecular modeling. Journal of Molecular Structure, 994(1), 163–169. Gonçalves, F. P., Martins, M. C., Junior, G. J. S., Lourenço, S. A., & Amorim, L. (2010). Postharvest control of brown rot and Rhizopus rot in plums and nectarines using carnauba wax. Postharvest Biology and Technology, 58(3), 211–217. Hofrichter, M. (2002). Review: lignin conversion by manganese peroxidase (MnP). Enzyme And Microbial Technology, 30(PII S0141-0229(01)00528-24), 454–466. Hwang, I., Sheen, J., & Müller, B. (2012). Cytokinin signaling networks. Annual Review of Plant Biology, 63, 353–380. Kachroo, A., & Robin, G. P. (2013). Systemic signaling during plant defense. Current Opinion in Plant Biology, 16(4), 527–533. Kim, H. J., & Miller, W. B. (2009). GA4+7 plus BA enhances postproduction quality in pot tulips. Postharvest Biology and Technology, 51(2), 272–277. Lee, B. R., Muneer, S., Jung, W. J., Avice, J. C., Ourry, A., & Kim, T. H. (2012). Mycorrhizal colonization alleviates drought-induced oxidative damage and lignification in the leaves of drought-stressed perennial ryegrass (Lolium perenne). Physiologia Plantarum, 145(3), 440–449. Li, L., & Steffens, J. C. (2002). Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta, 215(2), 239–247. Liu, H., Jiang, W., Bi, Y., & Luo, Y. (2005). Postharvest BTH treatment induces resistance of peach (Prunus persica L. cv. Jiubao) fruit to infection by Penicillium expansum and enhances activity of fruit defense mechanisms. Postharvest Biology and Technology, 35(3), 263–269. Lurie, S., Fallik, E., Handros, A., & Shapira, R. (1997). The possible involvement of peroxidase in resistance to Botrytis cinerea in heat treated tomato fruit. Physiological and Molecular Plant Pathology, 50(3), 141–149. Lutts, S., Kinet, J. M., & Bouharmont, J. (1996). NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Annals of Botany, 78(3), 389–398. Ma, Z., Yang, L., Yan, H., Kennedy, J. F., & Meng, X. (2013). Chitosan and oligochitosan enhance the resistance of peach fruit to brown rot. Carbohydrate Polymers, 94(1), 272–277. Mari, M., Martini, C., Guidarelli, M., & Neri, F. (2012). Postharvest biocontrol of Monilinia laxa, Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains. Biological Control, 60(2), 132–140.
Y. Zhang et al. / Food Chemistry 187 (2015) 210–217 Massolo, J. F., Lemoine, M. L., Chaves, A. R., Concellón, A., & Vicente, A. R. (2014). Benzylaminopurine (BAP) treatments delay cell wall degradation and softening, improving quality maintenance of refrigerated summer squash. Postharvest Biology and Technology, 93, 122–129. Mayer, A. M. (2006). Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry, 67(21), 2318–2331. Rungjindamai, N., Jeffries, P., & Xu, X. M. (2014). Epidemiology and management of brown rot on stone fruit caused by Monilinia laxa. European Journal of Plant Pathology, 1–17. Sholberg, A. P., & Kappel, F. (2008). Integrated management of stone fruit diseases. Integrated management of diseases caused by fungi, phytoplasma and bacteria (pp. 3–25). Netherlands: Springer. Spadoni, A., Neri, F., Bertolini, P., & Mari, M. (2013). Control of Monilinia rots on fruit naturally infected by hot water treatment in commercial trials. Postharvest Biology and Technology, 86, 280–284. Vicente, A. R., Saladie, M., Rose, J. K., & Labavitch, J. M. (2007). The linkage between cell wall metabolism and fruit softening: Looking to the future. Journal of the Science of Food and Agriculture, 87(8), 1435–1448. Vilanova, L., Viñas, I., Torres, R., Usall, J., Buron-Moles, G., & Teixidó, N. (2014). Increasing maturity reduces wound response and lignification processes against Penicillium expansum (pathogen) and Penicillium digitatum (non-host pathogen) infection in apples. Postharvest Biology and Technology, 88, 54–60. Wei, Y., & Ye, X. (2011). Effect of 6-benzylaminopurine combined with ultrasound as pre-treatment on quality and enzyme activity of green asparagus. Journal of Food Processing and Preservation, 35(5), 587–595.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
6-Benzylaminopurine inhibits growth of Monilinia fructicola and induces defense-related mechanism in peach fruit Yangyang Zhang, Lizhen Zeng, Jiali Yang, Xiaodong Zheng, Ting Yu ⇑ College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food Technology and Equipment, Zhejiang University, Hangzhou 310058, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 2 December 2014 Received in revised form 5 April 2015 Accepted 21 April 2015 Available online 23 April 2015 Chemical compounds studied in this article: 6-Benzylaminopurine (PubChem CID: 62389) Superoxide dismutase (PubChem CID: 5359597) Malondialdehyde (PubChem CID: 10964)
a b s t r a c t This study demonstrated the inhibitory effect of 6-benzylaminopurine (BAP), the first generation synthetic cytokinin, on the invasion of Monilinia fructicola in peach fruit and the possible mechanism involved for the first time. Our results suggested that BAP treatment had a 63% lower disease incidence and approximately 10 times lower lesion diameter compared to the control throughout the incubation period. In vitro BAP showed a direct inhibitory effect on M. fructicola spore germination. BAP could prevent fruit texture deterioration and protect the cell membrane from oxidative stress, while no adverse effects were observed on fruit quality maintenance. Analysis of defense-related enzymes activities indicated that the use of BAP induced higher specific polyphenol oxidase and peroxidase activities which triggered stronger host defensive responses. Thus, our results verified the proposed mechanism of BAP in controlling M. fructicola by direct inhibitory effect, delay peach senescence and activation of defensive enzymes. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: 6-Benzylaminopurine (BAP) Monilinia fructicola Peach Postharvest
1. Introduction Peaches are one of the most important fruit crops cultivated in east and north areas of China with an estimated annual production approximate 12 million metric tons in 2012 (Food, 2012). Brown rot, caused by Monilinia fructicola, is a serious bacterial disease that affects peaches quality from blossom period to harvest storage, with broad devastating dissemination causing production loss estimated at 20% or more (Zhu, Chen, Luo, & Guo, 2005). Due to the potential hazard of synthetic fungicides on human being’s health and the environment, none of the fungicide spray programs are authorized for postharvest control of M. fructicola. Hence there is a growing interest in alternative strategies for M. fructicola inhibition (Casals et al., 2012). Within these alternatives there are three main fields (physical control, biological agents and chemicals). The use of cold storage, radio frequency and hot water treatment has been proposed to ⇑ Corresponding author at: 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China. E-mail address: [email protected] (T. Yu). http://dx.doi.org/10.1016/j.foodchem.2015.04.100 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
control brown rot on peaches (Spadoni, Neri, Bertolini, & Mari, 2013). Some biological agents have shown satisfactory efficacy for M. fructicola control, such as Bacillus subtilis CPA-8 and Aureobasidium pullulans strains (Mari, Martini, Guidarelli, & Neri, 2012). Nevertheless, these methods have some drawbacks and limitations in commercial fruit production. Physical treatments need large equipment to process fruit and may damage sensory quality, while biocontrol agents are limited by the low effectiveness. (Rungjindamai, Jeffries, & Xu, 2014; Sholberg & Kappel, 2008). Applications of chemicals with non-toxicity provide effective inhibition against postharvest decay and high value of commercial promotion as well. Several natural chemical compounds, such as chitosan, tea polyphenol and carnauba wax (Gonçalves, Martins, Junior, Lourenço, & Amorim, 2010; Ma, Yang, Yan, Kennedy, & Meng, 2013), have demonstrated direct inhibitory actions on M. fructicola growth. Therefore, non-toxic chemicals seem to be the promising alternatives to the synthetic fungicides. Cytokinins are involved in various aspects of plant biological processes, ranging from organ formation and apical dominance to leaf senescence (Hwang, Sheen, & Müller, 2012). 6Benzylaminopurine (BAP), the first generation synthetic cytokinin,
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plays a significant role in plant cell division and enhancing chilling tolerance (Chen & Yang, 2013). Moreover, BAP has been approved as biopesticide in postharvest practices by the United Stated Environmental Protection Agency (US-EPA). In harvest commodities, spray application of BAP is involved in extending the vase life of pot tulips and improving quality maintenance of summer squash (Kim & Miller, 2009). Direct evidences concerning the applications of cytokinins on postharvest pathogens control are scarce, little information evaluates the efficiency of BAP alone on brown rot control in peach fruit. The objectives of this study were to determine the efficiency of different concentrations of BAP in controlling peach brown rot caused by M. fructicola during the postharvest storage and the possible mechanisms involved.
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2.4. Effect of time between BAP treatment and pathogen inoculation on inhibition of M. fructicola To assay the effect of time between BAP treatment and pathogen inoculation on inhibition of M. fructicola, peach fruit were washed, sterilized and wounded following the method described above. Fifty microliters of 500 mg/l BAP was added into the wounds of peach fruit. After a series time (0, 6, 12 and 24 h) of BAP treatment, thirty microliters of M. fructicola spore suspension (1 104 spores ml1) was inoculated into each wound as well. Fruit without BAP treatment acted as the inoculated control. Then fruit were stored in the thermostatic cultivation room at 25 °C for recording the disease incidence and lesion diameter. All treatments were conducted with three replicates and ten fruit per replicate, which were repeated twice.
2. Materials and methods
2.5. Effect of BAP on spore germination in PDB
2.1. Fruit and chemicals
The effect of BAP on spore germination of M. fructicola was assayed in PDB. Pure colonies of M. fructicola were used to prepare suspensions concentration of 1 105 spores ml1. One fifth of a milliliter of each suspension blended with 1 ml BAP at final concentrations of 1, 10, 100, 200, 500 and 1000 mg/l were distributed into a test tube containing 0.8 ml PDB, sterile distilled water was used as control. All test tubes were incubated at 25 °C for 24 h in the dark. Approximately 100 spores of M. fructicola were measured randomly in the microscopic visual field for germination rate and germ tube length. Each treatment included three test tubes randomly within three replicates.
Peach (Prunus persica L. Batsch. cv. Hanlumi) were harvested at the commercial mature stage in Shandong Province, China and sorted based on the size and the absence of physical injuries or disease infection. Prior to treatments, fruit were sterilized in a water solution of 0.1% sodium hypochlorite for 2 min, rinsed with tap water completely and then desiccated at room temperature. An isolate of M. fructicola was obtained from decayed peach fruit and cultured on potato dextrose agar (PDA) medium at 25 °C in the dark. Sporangiospore suspension was prepared from the flooded 7-days-old cultures and suspended in sterile distilled water. Afterwards the spore concentration was observed by the means of a hemocytometer and adjusted to 1 104 spores ml1 with sterile distilled water. BAP was purchased from Sinopharm Chemical Reagent Company (Beijing, China). The stock solution of BAP was diluted to a series (1, 10, 100, 200, 500 and 1000 mg/l). 2.2. Effect of BAP on inhibition of M. fructicola in vivo Each peach fruit was wounded softly with the sterile punch in the equatorial region to form a hole (5 mm deep and 5 mm in diameter). Fifty microliter of BAP at different concentration was added to the wound separately in each treatment: BAP at different concentrations (100, 200, 500, 1000 mg/l) and sterile distilled water served as control. Thirty microliters of M. fructicola spore suspensions at 1 104 spores ml1 were inoculated into the wounds of all peach fruit 1 h later. Fruit were placed into plastic trays and packaged with preservative film in the thermostatic cultivation room for incubation at 25 °C with high relative humidity (90–95%). The number of infected peaches was monitored daily. Three replicates of 20 peaches were selected as a unit for each treatment, the disease incidence and lesion diameter of total peaches were recorded for statistical analysis, respectively. 2.3. Effect of BAP on inhibition of M. fructicola at low temperature Peach fruit were wounded in each fruit equatorial region as above. Each wound was treated with fifty microliter of BAP in a series of concentration: 100, 500 and 1000 mg/l; wounds treated with equal volumes of sterile distilled water served as controls. Twenty peach fruits were packaged as a group and thirty microliters of M. fructicola (1 104 spores mL1) spore suspensions were pipetted into the wounds. Afterwards all fruit were incubated at 4 °C in refrigerated cabinet. All treatments were conducted with three groups as replicate.
2.6. Effect of BAP on survival of M. fructicola on PDA M. fructicola spore suspension was mixed with serial concentrations of BAP (0, 1, 10, 100, 200, 500 and 1000 mg/l) in order to adjust the final concentration to 100 spores ml1. The mixed suspensions were poured onto petri dishes (60 mm in diameter) with 15 ml PDA, respectively. Finally colonies per plate were calculated after 72 h incubation at 25 °C. Three petri dishes were chosen randomly as a replicate and each treatment included three replicates. 2.7. Effect of BAP on defense-relate enzymes activities in vivo For the enzyme assay, fifty microliters of 500 mg/l BAP was added to each wound and the same dose of sterile distilled water was regarded as control. The fruit tissues near the wounds were sampled after a series of time interval (0, 6, 12, 24, 36 and 48 h) and frozen in the liquid nitrogen immediately. Each sample treatment made up of three fruit tissues was prepared for the assay of enzyme activities. Each frozen tissue sample (0.2 g) was homogenized with 1 ml of sodium phosphate buffer (50 mM, pH 7.8) comprising 1.33 mM Ethylenediaminetetraacetic acid and 1% (w/v) polyvinyl-pyrrolidone (PVP) and ground at 4 °C. The homogenate was centrifuged at 12,000g for 10 min, then the extracts obtained were used for the enzyme assay of superoxide dismutase (SOD), polyphenol oxidase (PPO) and peroxidase (POD) at 4 °C. SOD activity was determined by the method of using colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), following the manufacturers specifications. Results of SOD activity was expressed as units per lg of protein. PPO activity was measured by using pyrocatechol as a substrate following the method of Aquino-Bolaños and Mercado-Silva (2004). Fifty microliters crude enzyme was blended with 250 ll 50 mM sodium phosphate buffer (pH 6.4) containing 10 mM pyrocatechol, the reaction was incubated for 1 min at 30 °C, then the absorbance of the supernatant was recorded once 30 s for 10 times
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at 398 nm using a spectra max plus 384 (Molecular Devices Limited, USA). One unit of PPO activity was defined as the amount of enzyme extracts producing an increase of A398 nm by 0.01 in 1 min and the activity was expressed as units per lg of protein. POD activity determination was operated according to the method of Lurie, Fallik, Handros, and Shapira (1997). The reaction mixture was made up of 220 ll of 0.3% (v/v) guaiacol and 20 ll of crude enzyme extracts. The reaction was started by the addition of 60 ll of 0.3% (w/v) H2O2, later the change of absorbance at 460 nm was measured once 30 s during the whole process of 5 min. One unit of POD activity is defined as the amount of enzyme required to cause an increase in absorbance of 0.01 at 470 nm per minute, the enzyme activity was expressed as units per lg of protein. MDA content was measured according to the method of Du and Bramlage (1992), with some modification. A fifth of one gram of frozen tissue was ground with 1 ml 10% (w/v) trichloroacetic acid buffer and the homogenate was centrifuged at 12,000g for 10 min at 4 °C. Enzyme preparation containing 700 ll of crude enzyme and the same volume of 0.6%(w/v) thiobarbituric acid was incubated for 10 min at 100 °C. After that the reaction mixture was cooled in ice immediately and centrifuged at 4000g for 15 min at 4 °C, the volume of supernatant was measured also. The absorbance of reaction mixture was measured at 532, 600 and 450 nm, respectively, while 0.6% (w/v) TBA was regarded as blank. MDA was calculated according to the formula:
C MDA ðlmol=lg proteinÞ ¼ ð6:45 ðA532 A600 Þ 0:56 A450 Þ ðextract volumeÞ=ðprotein weightÞ Protein content of each enzyme assay was determined according to Bradford (1976) with bovine serum albumin (Sigma Chemicals Co., St. Louis, USA) as standard. Each treatment contained three replicates and the experiment was repeated twice.
collected from opposite regions at the equator of fruit were shaped into discs of 8 mm thick and 2 g weight. Then samples were immersed in 5 ml of deionized water for 10 min to fill the fruit cells with deionized water, subsequently samples were placed in the vacuum desiccator at 0.06 MPa for 10 min. The reaction mixture was incubated on a rotary shaker at 150g for 1 h at room temperature. After briefly vortexing, electrolyte conductivity was determined with a conductivity meter (Model DDS-11A, Shanghai Scientific Instruments) as initial (c1), afterwards samples were treated with boiling water heating for 15 min and the electrolyte conductivity were second recorded (c0) after cooled down to room temperature. The electrolyte conductivity was defined as follows:
Electrolyte conductivity ¼ c1 =c0 100% To evaluate the content of phenolic, flavonoids and anthocyanin, fruit samples were prepared as described above for determinations. The supernatant was extracted from 2 g of frozen tissue with 20 ml ice-cold 1% HCl–methanol solution and then centrifuged at 15,000g for 15 min at 4 °C. 1% HCl–methanol solution was regarded as blank, accordingly phenolic content was defined as OD280/g, flavonoids content was expressed as OD325/g and anthocyanin content equaled to (OD530 OD600)/g.
2.9. Data analysis All statistical analyses were performed with SPSS 19.0 statistical software by one-way analysis of variance and Duncan’s multiple range tests. When the number of means in each group is two, the independent samples Tukey’s test is applied for means separation. p-Values of different treatments less than 0.05 were considered to be significant.
2.8. Effect of BAP on peach quality
3. Results
The effect of BAP on fruit quality was investigated on peach fruit. Fruit were immersed in solutions of sterile distilled water (control) and 500 mg/l BAP, respectively for 10 min and then incubated in cold storage cabinet at 4 °C. Fruit quality parameters in each treatment during storage was measured by monitoring changes of the fruit at 16 days. At the sampling date, peach from both control and BAP-treated groups were taken for quality evaluation (firmness, titratable acidity and membrane permeability). The determination of fruit firmness was conducted according to the method of Cao, Jiang, and Zhao (2011, chap. 2). The samples were peeled to 1 mm, fruit firmness was made at two equidistant points on the equator of each fruit with a hand-held penetrometer (GY-2, Fruit Firmness Tester, Zhejiang, China). The average of those two results was considered as one replicate. Ten fruit per treatment were determined and data are expressed in megapascal (MPa). Titratable acidity (TA) content was assayed by means of Cao et al. (2011, chap. 2). Samples of the mashed fruit (5.0 g) were centrifuged by 4000g for 10 min and the supernatants were used for titrating to pH 8.1 with 0.01 mol l1 NaOH, phenolphthalein functioned as the indicator. TA content was calculated as percentage of malic acid which is multiplied by the conversion factor of organic acid concentration per gram of fresh mass. Each treatment was consisted of three replicates and per replicate was pooled from three wounds that were collected from three fruit, the experiment was performed twice. For the membrane permeability assay, membrane permeability was evaluated on the basis of electrolyte leakage according to the method of Lutts, Kinet, and Bouharmont (1996) with slight modification. The percentage loss of electrolyte was calculated. Samples
3.1. Effect of BAP on inhibition of M. fructicola in vivo Fruit deterioration was significantly reduced by BAP treatments in vivo in a concentration dependent manner (Fig. 1A and B). After 60 h incubation, the disease incidence reached values of 77.3% and 2.6% in control and 100 mg/l BAP-treated fruit. BAP at concentrations of 500 and 1000 mg/l significantly suppressed disease incidence throughout incubation period and resulted in 49.4% and 38.7% at 108 h, respectively. Average lesion diameter values of those treatments were generally 10 times lower than that of control. When the concentration of BAP treatment increased from 500 to 1000 mg/l, no significant differences were observed in the disease incidence and average lesion diameter.
3.2. Effect of BAP on inhibition of M. fructicola at low temperature Fig. 1C showed the brown rot incidence of different concentrations of BAP at 4 °C storage. The wounds inoculated with BAP were effective in inhibiting the M. fructicola infection. BAP at lower-dose treatment was not sufficient to control rot occurrence at 228 h. While no appearance of brown rot was detected in 500 mg/l BAP treatment until 336 h and the peak of disease incidence reached 33.3% at the end period of storage. Average lesion diameters were significantly lower than those in control treatment (Fig. 1D). Disease incidence of 1000 mg/l BAP treatment resembled that of 500 mg/l BAP treatment but peaked at 46.7%. Low temperature storage could extend the incubation time approximately 4-fold than that in 25 °C storage, whereas it could provide enduring protection of peach fruit from the pathogen attack.
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Fig. 1. Inhibitory effect of 6-benzylaminopurine (BAP) at different concentrations on growth of M. fructicola in peach fruit. Disease incidence (A) and average lesion diameter (B) of peach fruit infected by M. fructicola were investigated for 108 h at 25 °C. Serial concentrations of BAP at 100, 200, 500 and 1000 mg/l were pipetted into fruit wounds to suppress the growth of M. fructicola (1 104 spores ml1) in vivo, using sterile distilled water as a control. Disease incidence (C) and average lesion diameter (D) of peach fruit infected by M. fructicola were recorded throughout incubation period at 4 °C. Fruit wounds were treated with sterile distilled water (as control) and BAP at various concentrations: 100, 500 and 1000 mg/l to inhibit the growth of M. fructicola (1 104 spores ml1). Vertical bars represent standard errors of three replicates.
3.3. Effect of treatment time interval on inhibition of M. fructicola Results above had indicated that peach fruit treated with 500 mg/l BAP showed a remarkable suppression on M. fructicola growth (Fig. 1). The percentage of infected fruit decreased when the treatment time interval between BAP treatment and inoculation of M. fructicola was 12 h or longer (Fig. 2A). After 24 h incubation, disease incidence reached the values of 18.7% and 25.3% in 12 and 24 h treatment, respectively, which were generally lower than
that in 0 h treatment at the value of 78.8%. There was no significant difference in disease incidence and average lesion diameter between 12 and 24 h treatment time interval. 3.4. Effect of BAP on spore germination of M. fructicola in PDB BAP treatments suppressed the spore germination of M. fructicola in a dose-dependent manner (Fig. 3A). Percentage of spore germination was 93.3% and 81.2% for control and 10 mg/l BAP
Fig. 2. Inhibitory effect of time interval between 6-benzylaminopurine (BAP) treatment and inoculation of M. fructicola in peach fruit at 25 °C. Thirty microliters of M. fructicola suspension (1 104 spores ml1) was inoculated at a serial time interval (0, 6, 12 and 24 h) after fifty microliters of 500 mg/l BAP was added into fruit wounds. Disease incidence (A) and average lesion diameter (B) of peach fruit infected by M. fructicola were investigated after 84 h incubation. Standard errors of three replicates were described as vertical bars.
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Fig. 3. In vitro antifungal activity of 6-benzylaminopurine (BAP) against M. fructicola throughout incubation at 25 °C. Effect of BAP at different concentrations (10, 100, 200, 500 and 1000 mg/l) on spore germination of M. fructicola in peach fruit after incubation at 25 °C in PDB for 24 h (A). Effect of BAP at different concentrations (1, 10, 100, 200, 500 and 1000 mg/l) on survival of M. fructicola 72 h after inoculation on PDA (B). Vertical bars represent standard deviations of the means. Significantly different values are identified with different letters according to Duncan’s multiple range test at p < 0.05.
treatment, respectively. When the concentration of BAP increased to 200 mg/l, spore germination (Fig. 3A) and germ tube elongation (not shown) of M. fructicola was significantly inhibited. Moreover, the germination rate in 1000 mg/l BAP treatment was 70.2% lower than that of control, indicating that BAP retarded the growth of M. fructicola spores in vitro. 3.5. Effect of BAP on survival of M. fructicola on PDA Fig. 3B illustrated the inhibitory effect of BAP on colony growth of M. fructicola on PDA. Suppression on M. fructicola colony depended on the concentration of BAP treatment: the higher concentration, the stronger inhibition. BAP at concentration of 100 mg/l exhibited significantly colony reduction (p < 0.05) compared with that in control. When the application of BAP concentration increased to 1000 mg/l, the value of average colony number was merely 3.7 colonies per plate. In all BAP treatments, values of average colony number were generally lower than the control at 56.9, which demonstrated that BAP was assuredly lethal to spore germination of M. fructicola in vitro. 3.6. Effect of BAP on defense-related enzymes activities Fig. 4 indicated the change pattern of defense-related enzymes relying response to BAP. The activity of SOD in BAP treatment increased continuously up to 48 h, an increment in SOD activity was detected at 24 h (p < 0.05), which was approximately 4-fold higher than that in control (Fig. 4A). SOD activity in control was generally lower than that in BAP-treated samples throughout incubation period. PPO activity in BAP treatment peaked at 12 h and then declined to 48 h (Fig. 4B). In contrast, control samples showed relatively stable in PPO activity. In BAP treatments, POD activity rapidly started to increase at 6 h and reached a peak at 12 h, in correlation with the induced PPO activity in peach (Fig. 4C), while little change of POD activity was observed in the control. In all samples, the accumulation of MDA started to increase at 6 h and continuously increased during incubation (Fig. 4D), however the application of BAP decreased MDA accumulation to a lesser extent than that of control samples. 3.7. Effect of BAP on peach quality The quality parameters were measured after 16 days storage at 4 °C. Fruit firmness is the most significant characteristic reflecting sensory quality of peach fruit (Table 1). Fruit firmness decreased
significantly to 9.38 MPa in the control, which showed up a little soft rotten. Firmness of BAP-treated fruit was 13.8 MPa showing no marked change during the storage. Moreover, no significant differences of firmness were recorded in BAP-treated peach throughout storage. Level of titratable acidity decreased in both control and BAP-treated fruit during refrigerate storage, but no differences were found between both groups of peach. An increase in cell membrane permeability indicated the permeation of cytoplasm and cell membrane damage, the values of cell membrane permeability were 59.2% and 48.3% for control and 500 mg/l BAP treatment, respectively, although an increment in BAP-treated fruit was observed after 16 days, the cell membrane permeability delayed by BAP was still significantly lower than that of the control. Change of total phenolic content in the peach fruit are shown in Table 2, an increasing level of total phenolic was found at the end of storage period, but no differences were found between control and BAP-treated fruit. The same trends were also observed in flavonoids and anthocyanins accumulation, the content of flavonoids and anthocyanins showed a slight increment in all treatments but was close to that in initial treatment, there were no statistical differences between control and BAP treatment. The present results indicated that BAP treatment could prevent fruit firmness deterioration and delay cell membrane permeability damage, while no adverse effects were observed on titratable acidity and total phenolic accumulation.
4. Discussion Previous studies had reported that cytokinins integrated with other measures on fruit were effective in reducing postharvest pathogen infection by inhibitory effect against postharvest pathogen and inducing defense system in plant host (Yu, Wang, Yin, Feng, & Zheng, 2008). Results from this study indicated that BAP directly inhibited the growth of M. fructicola in vivo and it obtained satisfactory control of M. fructicola throughout the incubation when the concentration increased to 500 mg/l or above (Fig. 1A and B), this data was similar to an earlier report by Zheng, Yu, Chen, Huang, and Wu (2007), where they found the application of BAP was sufficient to suppress Penicillium expansum infection in the early stage. Moreover, cold storage could greatly prolong the incubation time 4-fold longer than that at the optimal temperature of 25 °C (Fig. 1C and D). To our knowledge, this is the first work showing that BAP can effectively suppress M. fructicola infection in peach fruit. Homoplastically, results of assays in vitro indicated the direct inhibitory effect of BAP on spore germination
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Fig. 4. Effect of 6-benzylaminopurine (BAP) on the activities of superoxide dismutase (SOD) (A), polyphenol oxidase (PPO) (B), peroxidase (POD) (C) and malondialdehyde (MDA) content (D) in peach fruit. Peach fruit treated with fifty microliters of 500 mg/l BAP and aliquot of sterile distilled water (as control) were sampled at a series of time point (0, 6, 12, 24, 36 and 48 h) after inoculation. Asterisks indicate significant differences between BAP treatment and control sample. Vertical bars represent standard deviations of the means. Significantly different values are identified with different letters according to Duncan’s multiple range test at p < 0.05.
Table 1 Effect of 6-benzylaminopurine on peach fruit quality parameters stored after 16 days at 4 °C. Treatment
Firmness (MPa)
Titratable acidity (%)
Membrane permeability (%)
Initial 16 days control 16 days BAP
13.94 ± 0.97a 9.38 ± 2.17b
0.24 ± 0.01a 0.16 ± 0.02b
34.79 ± 1.37c 59.17 ± 4.29a
13.80 ± 3.05a
0.16 ± 0.02b
48.29 ± 3.12bc
Peach fruit were immersed in sterile distilled water and the solutions of 500 mg/l BAP for 10 min and air-dried subsequently. Samples were obtained after 16 days incubation at 4 °C. Values identified with the different letter in columns show significant difference on the basis of Duncan’s multiple range test (p < 0.05), each value represents a mean of three replicates (n = 3).
Table 2 Effect of 6-benzylaminopurine on peach fruit quality parameters (total phenolic, flavonoids and anthocyanins) after 16 days incubation at 4 °C. Treatment
Total phenolic (DOD/g)
Flavonoids (DOD/g)
Anthocyanins (DOD/g)
Initial 16 days control 16 days BAP
7.84 ± 0.48a 8.50 ± 0.55a
8.18 ± 0.48a 8.99 ± 0.60a
0.015 ± 0.002a 0.018 ± 0.004a
9.33 ± 0.50a
9.79 ± 0.49a
0.015 ± 0.000a
Peach fruit were immersed in sterile distilled water and the solutions of 500 mg/l BAP for 10 min and air-dried subsequently. Samples were obtained after 16 days incubation at 4 °C. Values identified with the different letter in columns show significant difference on the basis of Duncan’s multiple range test (p < 0.05), each value represents a mean of three replicates (n = 3).
of M. fructicola in vitro. BAP has been considered as nontoxic substance for the postharvest applications by US-EPA. Therefore BAP, due to its non phytotoxicity and antimicrobial activity, supports the possibility of a promising alternative strategy for postharvest brown rot management. Furthermore, BAP treatment was also found to delay softening in peach fruit efficaciously without adverse effects on quality parameters of peaches. This observation may be associated with the tissue susceptibility to dehydration and disassembly of cell wall polysaccharides (Vicente, Saladie, Rose, & Labavitch, 2007). Consistent with this possibility, Massolo, Lemoine, Chaves, Concellón, and Vicente (2014) reported that effect of BAP on firmness maintenance might be attribute to cell wall reinforce. BAP is also an inhibitor of respiratory kinase in plants and increases postharvest life of green vegetables in the field of agriculture (Ge et al., 2011), the firmness maintenance demonstrated that delay of peach senescence, corresponding to the application of BAP, resulted in an enhanced protection against pathogen invasion, which provided a hypothesis to explain the role of BAP in plant defense system. Interestingly, our results showed that BAP was more effective against M. fructicola infection when the time interval between BAP treatment and pathogen inoculation was up to 24 h in fruit wounds. This observation suggested that more efficient inhibition of brown rot in peach may be associated with host-mediated resistance induced by BAP. In general, plants can acquire enhanced resistance to pathogen infection when response to triggering factor such as chemical inducers. Although, not much is known about the molecular mechanism underlying this process, it does require a
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time frame within which it occurs from signal generation to robust activation of host defense in the systemic tissues (Kachroo & Robin, 2013). For example, previous reports indicated that chitosan, a chemical elicitor, could directly inhibit the growth of M. fructicola in peach fruit, but also initiate defensive mechanisms against pathogenic attacks (Yang, Zhang, Bassett, & Meng, 2012). In addition, it was worth noting that when the time interval reached 24 h, disease incidence was approximately three times lower than that in 0 h treatment, which showed that the improvement of induced defense mechanism could be the major factor underlying the effect of BAP, direct inhibition on the growth of M. fructicola might be the rapid and more efficient measures. Recent findings had suggested that the mechanism of induced resistance was involved in the activation of defense enzymes, accumulation of antifungal compounds, pathogen-resistant protein, increasing of reactive oxygen species and lignification of epidermal cells. (Vilanova et al., 2014). Our results showed an increment in SOD activity was detected in BAP-treated peach, which was consistent with the results reported by Xu et al. (2011). SOD, identified as essential component in antioxidant defense, converts superoxide radicals into hydrogen peroxide and oxygen. Within a cell, SOD constitute the first line of detoxification of reactive oxygen species (ROS) to prevent the destruction of the cell membrane in pathogen invasion (Alscher, Erturk, & Heath, 2002). MDA has been recognized as a relevant product of polyunsaturated fatty acids, which reflects the oxidative damage of cell membrane under adversity environment (Del Rio, Stewart, & Pellegrini, 2005). Current study showed a decrement of MDA accumulation in peach tissues inoculated with BAP even though MDA level was not completely suppressed. This result was in accordance with a previous study by Xu et al. (2011), where they suggested BAP was useful to delay the MDA accumulation within the cell. Apart from MDA content, permeability of cell membrane could be potentially related to the cell membrane damage, it has been shown that BAP was effective in cell membrane protection (Zavaleta-Mancera et al., 2007). In the present study, this must be explained by a mechanism in which the high level of antioxidant enzymes induced by BAP and protection of cell from oxidative stress could delay plant host senescence in order to restrain infection by the pathogen (Xu et al., 2014). POD and PPO are known to play a key role in fruit host defense mechanism (Liu, Jiang, Bi, & Luo, 2005). POD activity was also associated with lignification resulting in reinforcement of cell walls warding off pathogen penetration (Hofrichter, 2002). Higher activity of POD was induced in wounds inoculated with BAP compared to the control treatment. Similarly, an increase in POD activity in apple fruit, as a result of gibberellic acid treatment, was found to enhance plant resistance to grey mould rots (Yu & Zheng, 2007). Our results indicated that an increment in PPO activity, detected after the application of BAP, might be associated with producing antimicrobial phenolic substances. A previous report by Wei and Ye (2011) supported this result, they demonstrated that activities of PPO and POD were enhanced in green asparagus treated with BAP. PPO is a ubiquitous copper-containing enzyme in plants and probably involves in catalyzing the oxygen-dependent oxidation of phenols to quinones (Mayer, 2006). Previous studies indicated that PPO was associated with the lignification of plant cells and suberin formation during the microbial invasion (Lee et al., 2012). While the PPO-overexpressing tomato plants exhibited a great increase in plant defense against Pseudomonas syringae in the infection process (Li & Steffens, 2002). These results suggested a positive correlation between the enhancement of POD and PPO activity and the stronger induced defense responses. In conclusion, results in this study demonstrated that BAP could directly inhibit the growth of M. fructicola and postpone the deterioration in postharvest peach fruit. The delay of fruit senescence and enhanced activities of defensive enzymes had improved fruit
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