Research Article Received: 23 January 2014
Revised: 21 July 2014
Accepted article published: 28 July 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6845
Biocontrol of post-harvest Alternaria alternata decay of cherry tomatoes with rhamnolipids and possible mechanisms of action Fujie Yan,a,b,c Shixiang Xu,a,b,c Jun Guo,a,b,c Qianru Chen,a,b,c Qin Mengd and Xiaodong Zhenga,b,c* Abstract BACKGROUND: Rhamnolipids were reported to have evident antifungal activity. The efficacy of rhamnolipids against Alternaria alternata and their possible mechanisms involved were investigated. RESULT: The decay incidences of A. alternata of cherry tomatoes (Lycopersicon esculentum) treated by rhamnolipids were significantly reduced. The in vitro assays showed that rhamnolipids inhibited fungal growth on solid medium and prevented spore germination and mycelium growth in liquid medium. In addition, the combination of rhamnolipids and essential oil had a synergistic effect leading to the decrease of fungicidal concentrations of laurel oil. Scanning electron microscopy and transmission electron microscopy observations of the pathogen revealed significant morphological and cell structural alterations in the hyphae. Compared to the control, the content of nucleic acid in supernatant of the suspension of A. alternata increased, while the content of DNA and protein of mycelium decreased, which was in agreement with electrolyte leakage experiments. CONCLUSION: Rhamnolipids could be an alternative to chemicals for controlling post-harvest phytopathogenic fungi on fruits and vegetables. © 2014 Society of Chemical Industry Keywords: rhamnolipids; Alternaria alternata; cherry tomatoes; biological control; mechanisms
INTRODUCTION Considerable post-harvest losses of fruits and vegetables are brought about by decay caused by fungal plant pathogens. Chemical synthetic fungicides are the primary means to control post-harvest diseases but their side-effects such as high residues, long degradation period, strong and acute toxicity, environmental problem and production of fungicide-resistance1,2 make people pay attention to their usage. It is quite apparent that effective, safe and eco-friendly strategies need to be developed for reducing post-harvest losses as well as to make up the shortages of synthetic fungicides. Biosurfactants produced by a variety of microorganisms show unique properties such as mild production conditions and antimicrobial activity,3,4 compared to their chemical counterparts.5,6 Rhamnolipids are formed by one or two rhamnose molecules linked to one or two fatty acids of saturated or unsaturated alkyl chain between C8 and C12 .7 They produced by the bacterium Pseudomonas aeruginosa and are known as efficient anionic biosurfactant molecules.8 They are also used for a wide range of industrial applications, especially in food, cosmetics and pharmaceutical formulations as well as in bioremediation of pollutants.9,10 Rhamnolipids are applicable to a variety of pathogenic microorganisms especially for Gram-positive bacteria,11 – 13 but it is difficult to correlate the bacteriotoxic activity to the properties of rhamnose or fatty acid. It seems that the antimicrobial J Sci Food Agric (2014)
effects result from inhibition of pathogens metabolism, damage of plasma membranes and induction of fruit resistance such as relevant gene expression improvement.14,15 Furthermore, a cell-free culture broth as a crude rhamnolipid product showed the same evident antifungal activity against plant pathogens compared to di-rhamnolipid and mono-rhamnolipid with high purity, which means this crude fermentation product could serve as a potential cost-effective and eco-friendly alternative to chemicals for control of post-harvest phytopathogenic fungi.16 However, using rhamnolipids to control post-harvest diseases of fruits and vegetables also lacks systematic research, especially
∗
Correspondence to: Xiaodong Zheng, Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People’s Republic of China. E-mail: [email protected]
a Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People’s Republic of China b Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, People’s Republic of China c Zhejiang Key laboratory for agro-food processing, Zhejiang University, Hangzhou 310058, People’s Republic of China d Department of Chemical Engineering and Biochemical Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China
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www.soci.org with regard to fungal infections and some possible antifungal mechanisms. The objectives of this study were to evaluate the inhibitory ability of rhamnolipids in vitro and in vivo against A. alternata, a saprophytic pathogen of cherry tomato,17 and to undertake a preliminarily probe into the antifungal mechanisms of rhamnolipids.
MATERIALS AND METHODS Rhamnolipids and essential oil Rhamnolipids obtained from Professor Meng (Chemical Technology Department, Zhejiang University, Hangzhou, China) were produced via fermentation of P. aeruginosa ZJU-211 and purified through ultra-filtration, concentration and separation. The substrates consisted of two types of rhamnolipids: mono- and di-rhamnolipids. Rhamnolipids solutions (100 mg mL−1 ) were prepared with sterile distilled water and diluted to certain concentrations before being used in experiments. Pure-grade essential oil of Laurus nobilis was produced from International Flavors and Fragrances Inc., Shanghai, China. The essential oil was stored in bottles at 4 ∘ C. Fungi and cultures A. alternata was obtained from IMCAS (Institute of Microbiology, Chinese Academy of Science, China) and maintained on the medium of the potato dextrose agar (PDA: extract of 200 mL of boiled potatoes, 20 g of dextrose and 800 mL of distilled water) at 28 ∘ C. Spore suspensions were prepared by flooding a 7-day-old sporulating culture of A. alternata with sterile distilled water and the spore concentration was counted with a haemocytometer.18 In vitro antifungal assay PDA was autoclaved and cooled in a water bath to 40 ∘ C. Rhamnolipids were mixed with sterile molten PDA to obtain the final concentrations of 0, 125, 250, 500 and 1000 μg mL−1 and media were poured into 90-mm Petri plates (15 mL per plate). A 6 mm plug cut from a 7-day-old culture of A. alternata growing on PDA was placed on the centre of each plate and then incubated at 28 ∘ C. Radial growth was measured after 2 days until control group had grown all over the plate. Inhibition (%) = (1 − treatment plaque radius/control plaque radius) × 100. Growth rate (mm day−1 ) = plaque radius of dayn − plaque radius of dayn−1 (n = 3, 4, 5, 6). Five replicates were used for each treatment and experiments were performed twice. Alternaria alternata growth and spore germination assays A spore suspension (100 μL; 105 spores mL−1 ) was dispensed in the flasks containing rhamnolipids at concentrations of 0, 1000 or 2000 μg mL−1 . Dry weight of fungus was measured after a 14 day incubation and results were average of three independent conical flasks for each treatment. For germination assays, rhamnolipids were added to a 10 mL glass tube containing 5 mL PDB to obtain the medium with their final concentrations of 0, 200, 400, 600, 800 and 1000 μg mL−1 respectively. Then a spore suspension (100 μL; 106 spores mL−1 ) was added to each tube. After 20 h of incubation at 28 ∘ C on a rotary shaker (200 × g), at least 100 spores per replicate were observed microscopically to determine germination rate.19 There were three replicates per treatment and the experiment was conducted twice.
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Efficacy of rhamnolipids on inhibition of Alternaria rot in wounded cherry tomatoes Cherry tomatoes without physical injuries and infections were randomly selected. The fruits were gently wounded (6 mm diameter and approximately 3 mm deep) using a sterile borer. Each wound was treated with a solution at 30 μL of rhamnolipids at the concentrations of 0, 500, 1000, 1500 and 2000 μg mL−1 , respectively. After 2 h, 10 μL of spore suspensions of A. alternata (104 spores mL−1 ) were inoculated into each wound. Then the cherry tomatoes were stored in enclosed trays to maintain a high relative humidity at 25 ∘ C. Disease incidence was observed daily after 2 days. There were three replicates per treatment with 20 fruit per replicate and the experiment was conducted twice. Efficacy of rhamnolipids with essential oil from Laurus nobilis in reducing Alternaria alternata Minimal inhibitory concentration and minimal fungicidal concentration Laurel oil was added to a 10 mL glass tube containing 5 mL PDB to obtain the final concentrations ranged from 62.5 to 2000 μg mL−1 with or without rhamnolipids at 500 μg mL−1 . Then a spore suspension of A. alternata (100 μL; 106 spores mL−1 ) was added to each tube for 24 h at 27 ∘ C on a rotary shaker (200 × g). After incubation period, MIC, defined as the lowest concentration of laurel oil that inhibits development of visible microbial growth on PDB was determined. To determine the MFC, subcultures were grown on inhibitor-free nutrient agar plates from each clear tube in the MIC test series, followed by incubation at 27 ∘ C for 48 h.20 In vivo assays Cherry tomatoes were treated as described above. Each wound was treated with 30 μL of (1) sterile distilled water as the control; (2) a solution of rhamnolipids at 500 μg mL−1 ; (3) a solution of laurel oil at 500 μg mL−1 ; and (4) a solution of laurel oil at 500 μg mL−1 with rhamnolipids at 500 μg mL−1 . After 2 h, 10 μL of spore suspensions of A. alternata (104 spores mL−1 ) were inoculated into each wound. Then the cherry tomatoes were stored in enclosed trays to maintain a high relative humidity at 25 ∘ C. Disease incidence was observed daily after 2 days. There were three replicates per treatment with 20 fruit per replicate and the experiment was conducted twice. Efficacy of rhamnolipids in cell permeability of Alternaria alternata Firstly, a 14-day culture of A. alternata in PDB with or without rhamnolipids at 27 ∘ C was centrifuged for the next three experiments. Electrolyte leakage After washing cultures of A. alternata with deionised water, 1 g of sample was transferred into a small beaker and immersed in 20 mL deionised water. Each treatment was divided into two groups. One was exhausted and deflated repeatedly in the vacuum drying oven at 30 ∘ C for 3 h. The other was heated in the boiling water bath for 30 min to make plasma membrane completely destroyed. The conductivity of both specimens was measured using a conductivity meter, and the electrolyte leakage was calculated as the ratio of the conductivities of both groups. Determination of A260 values Through the detection of absorbance at 260 nm, one could estimate the amount of DNA and RNA released from the cytoplasm.
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J Sci Food Agric (2014)
Biocontrol of A. alternata in cherry tomatoes
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Scanning electron microscopy and transmission electron microscopy A spore suspension (100 μL; 105 spores mL−1 ) was dispensed in the flasks with or without rhamnolipids (1000 μg mL−1 ). After 14 days of incubation at 27 ∘ C on a rotary shaker (200 × g), mycelia were excised from a conical flask for electron microscope sample preparation. Samples were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) overnight at 4 ∘ C, then fixed with 1% osmium tetroxide in phosphate buffer (pH 7.0) for 1 h and rinsed three times in phosphate buffer (pH 7.0). Each specimen was dehydrated by the ethanol solutions with concentrations of 50%, 70%, 80%, 90%, 95%, 100% in turn for 15 min respectively. For scanning electron microscopy (SEM), the specimens were placed in an ethanol and isoamyl acetate mixture for 30 min and transferred to absolute isoamyl acetate for 2 h. The processed samples were observed by SEM.23 For transmission electron microscopy (TEM), the specimens were placed in an absolute acetone for 20 min, in a 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 h, in a 1:3 mixture of absolute acetone and the final Spurr resin mixture for 3 h and in final Spurr resin mixture overnight. After being placed in capsules containing embedding medium and heated at 70 ∘ C for about 9 h, the specimen sections were stained by uranyl acetate and alkaline lead citrate for 15 min, respectively, and observed in TEM.24 Statistical analyses Statistical analyses of the data were performed with SPSS for Windows Version 11.5. One-way ANOVA analyses and Duncan’s multiple range test were used to detect statistical significance, and differences were considered significant when P < 0.05.
50
Inhibition (%)
Measurement of DNA and protein content Total DNA was extracted from 150 mg dry mycelium using the Cetyltrimethyl Ammonium Bromide (CTAB) method. For protein measurement, 150 mg of dry mycelium was ground in liquid nitrogen with a mortar and pestle and transferred to a centrifuge tube containing 2 mL pre-cooled sodium phosphate buffer (pH 7.8). The homogenates were centrifuged at 4 ∘ C at 4000 × g, and the supernatants were assayed for measuring protein content according to Bradford’s method.22 There were three replicates for each treatment and each experiment was repeated twice.
A 60 a
a
a
40
b
30 20 10
c
0
0
125 250 500 -1 Rhamnolipids (µg mL ) control
B Growth rate (mm/day)
The method was according to Hou et al.21 with modifications. The supernatants after centrifugation were read at 260 nm with a nucleic acid analyser.
125
250
1000
500
1000
7 a
6 5
a
b a
b ab
b b b
4 3
a
a a
bc bc
c c
c
b b b
2 1 0
3d
4d
5d
6d
Days
Figure 1. The inhibitory effects of rhamnolipids. (A) Inhibition ratios measured at 6 days. (B) The growth rate of A. alternata in different concentrations of rhamnolipids. Bars represent standard errors of five replicates. Different letter indicates significant differences (P < 0.05) according to the Duncan’s multiple range test.
RESULTS In vitro antifungal assay At the concentration of 125 μg mL−1 , rhamnolipids had a relatively low inhibitory effect on radial growth of A. alternata, with the inhibitory ratio of 26.64%, while increasing to 40.19% at the concentration of 250 μg mL−1 . However, the inhibitory effect was not continuously enhanced when the concentration was higher than 250 μg mL−1 (Fig. 1A). Interestingly, the growth rate significantly slowed down after treated with rhamnolipids at 250 μg mL−1 or above (Fig. 1B), indicating that relative high concentrations of rhamnolipids were effective for delaying fungal growth.
Alternaria alternata growth and spore germination assays Compared to the control, the weight of hyphae was significantly reduced for the treatments at 1000 and 2000 μg mL−1 (Table 1). Spore germination in PDB was strongly inhibited by rhamnolipids and inhibitory ratio increased as the concentration increased. In the control group, 80.6% spores germed and the germ tubes were long and entwined. However, the germination of 64% of spores was inhibited after treated with 400 μg mL−1 and even the quantity was up to 81.7% with rhamnolipids at 1000 μg mL−1 (Fig. 2A).
Table 1. Effect of rhamnolipids (RLs) at concentrations of 0, 1000, 2000 μg mL−1 on A. alternata mycelium in potato dextrose broth medium Concentration of RLs (μg mL−1 ) 0 1000 2000
Dry weight (mg) 409.0 ± 13.53a 108.3 ± 45.37b 97.7 ± 56.58b
Electrolyte leakage (%) 24.2 ± 5.38b 35.9 ± 2.79ab 41.3 ± 0.10a
Protein content of mycelium (mg g−1 ) 13.4 ± 2.80a 13.0 ± 3.68a 4.9 ± 3.31b
DNA content of mycelium (μg g−1 )
A260 value of supernatant
1423.5 ± 332.05a 637.5 ± 69.58b 627.4 ± 98.05b
0.95 ± 0.03b 1.22 ± 0.09b 2.45 ± 0.21a
Values are the mean of three replicates ± SE. Data followed by different letters are significantly different according to Duncan’s multiple range test (P < 0.05).
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Table 2. Antimicrobial activity against A. alternata of laurel oil alone and in the presence of rhamnolipids (RLs) (500 μg mL−1 ) Laurel oil (μg mL−1 )
Parameter MIC MFC
Laurel oil + RLs (μg mL−1 )
1000 1200
500 1000
Disease incidence (%)
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F Yan et al. 100
a
80
Spore germination (%)
a
80 70 60 50 40 30 20 10 0
c
40 20 0
Control
c
e
200
400
600
800
1000
-1
Disease incidence (%)
48h 100
m
60
60h
72h
m
x
80
x
xy
a
m
m
m
b
y
40
c
z cd
20
d
0 0
ELs
RLs+ELs
Treatments
of A. alternata treated with rhamnolipids (Table 1). All these experiments showed that cell permeability of A. alternata obviously increased after treating with high concentrations of rhamnolipids.
cd
d
Rhamnolipids (µg mL )
B
RLs
Figure 3. Effect of laurel oil (500 μg mL−1 ) with rhamnolipids (500 μg mL−1 ) on reduction of A. alternata in cherry tomato fruit. Bars represent standard errors of three replicates. Different letter indicates significant differences (P < 0.05) according to the Duncan’s multiple range test.
b
0
bc
60
MIC, minimal inhibitory concentration; MFC, minimal fungicidal concentration.
A 90
ab
500
1000
1500
2000
-1
Rhamnolipids (µg mL )
Figure 2. (A) Effect of different concentrations of rhamnolipids on spore germination of A. alternata. (B) Effect of rhamnolipids on reduction of A. alternata in cherry tomato fruit. Bars represent standard errors of three replicates. Different letter indicates significant differences (P < 0.05) according to the Duncan’s multiple range test.
In vivo antifungal assay The results showed that disease incidence decreased as the concentration of rhamnolipids increased (Fig. 2B). The percentage of infected fruits was reduced from 70% (control) to 17% (rhamnolipids at 2000 μg mL−1 ) after 60 h of storage. Antimicrobial activity of essential oil alone and in the presence of rhamnolipids Values of minimal inhibitory concentration and minimal fungicidal concentration According to the experimental data, both values of MIC and MFC represented in Table 2 were considerably lower in the presence of rhamnolipids compared with the control. In vivo assays Rhamnolipids or laurel oil at 500 μg mL−1 alone were not effective in inhibiting the A. alternata infections in cherry tomato fruit wounds. However, disease was significantly inhibited with combined treatment, which was better than the treatment with rhamnolipids or laurel oil alone. After 4 days of incubation, the combination treatment reduced the A. alternata incidence to 43% (Fig. 3). Efficacy of rhamnolipids on cell permeability of Alternaria alternata In comparison with the control, electrolyte leakage and values of A260 of the supernatants increased by 71% and 158%, while DNA and protein content in mycelium decreased, after the suspension
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Scanning electron microscopy and transmission electron microscopy Through observation of SEM, A. alternata with no treatment showed even thickness hyphae with normal shapes (Fig. 4A), while the rhamnolipids-treated hyphae exhibited bumpy surfaces and breakage (Fig. 4B). Through observation of TEM, cells with no treatment showed smooth and intact membranes, homogeneous cytoplasms and structured cell walls (Fig. 5A and C). However, after being treated with rhamnolipids at 1000 μg mL−1 , cells exhibited membrane distortion and unclear cell wall structures as well as much smaller size (Fig. 5B and D).
DISCUSSION Rhamnolipids have an evident inhibitory effect on A. alternata, a type of main pathogen that will cause post-harvest diseases of cherry tomato. As the concentrations increased, the fungicidal effect became more remarkable within a certain period of time. However, when concentrations were higher than 3000 μg mL−1 (data not shown), the effect declined instead of being enhanced, and cherry tomato peel was wrinkled and shrank. The possible reason is that the fruit’s ability to resist fungal infection declines because of cell injury when treated with high concentrations of rhamnolipids. Therefore, it is important to choose a suitable concentration of rhamnolipids to apply to post-harvest preservation to make sure that fruit quality is maintained and there is an inhibitory effect. Additionally, as a kind of mild fungistat, rhamnolipids could only inhibit the growth of fungus rather than kill it, which could explain why all treatments had no significant difference on the in vivo assays after 72 h of incubation compared with the control. Undoubtedly, it is necessary to combine rhamnolipids with other safe material with much better effect of fungal inhibition, which can make up the defect that rhamnolipids alone cannot be used for fruit preservation for a long time. Essential oils extracted from plants and natural substances showed an excellent ability to control post-harvest pathogens and diseases in previous studies,23,25 but their inhibitory ability was not stable. They also caused severe fruit injury and a decline in disease resistance if the inhibitory concentration was a little higher. During the present work, we have found that rhamnolipids, as a type of microbial fermentation product, have a mildly inhibitory effect, but when they were combined with laurel oil we have observed a decrease of fungicidal concentration and a better effect on reduction of A. alternata in cherry tomato than the laurel oil alone.
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Biocontrol of A. alternata in cherry tomatoes
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A
B
Figure 4. Scanning electron microscopy photomicrographs of (A) the untreated mycelia of A. alternata (magnification, ×3000); (B) treated with 1000 μg mL−1 rhamnolipids (magnification, ×3000).
A
B
C
D
Figure 5. Transmission electron microscopy photomicrographs of (A) the untreated cell (magnification, ×30 000); (B) treated with 1000 μg mL−1 rhamnolipids (magnification, ×30 000); (C) the untreated cell (magnification, ×50 000); (D) treated with 1000 μg mL−1 rhamnolipids (magnification, ×50 000).
Mechanisms involved in the rhamnolipids inhibition of pathogenic bacteria and fungi have been mainly associated with damage of the microbial cell membrane, reduction of the spore movement ability and spore collapse.26 Moreover, a combination of rhamnolipids and thiosulfonic esters showed a synergisitic effect leading to a decrease of bacterial and fungicidal concentrations of ethyltiosulfonate (ETS) and methyltiosulfonate (MTS). Sotirova et al.20 found rhamnolipids would change the lipid composition of B. subtilis membrane as a result of increasing the content of cardiolipin, a kind of negatively charged phospholipid, and leading to enhancing membrane susceptibility to thiosulfonic esters. Results of our experiments regarding the combination of rhamnolipids and essential oil were similar to their research. We suppose that an interaction of rhamnolipids and plasma membrane of A. alternata allows essential oils to penetrate into cytoplasm more easily so they can exert a greater inhibitory effect. Rhamnolipids have a great inhibitory effect on breeding and growth of fungi by inhibiting their spore germination. A large quantity of hyphae and black spores could be observed in the control group, while the liquid culture with rhamnolipids turned J Sci Food Agric (2014)
to light yellow or even white. Formation and extension of germ tubes played a pivotal role in the pathogenicity of pathogen fungi27 – 29 and lengths of germ tubes were effectively suppressed by high concentrations of rhamnolipids. Obviously, the results of our experiment were consistent with the results reported by Kim et al.26 Mycelium plays an important role in pathogen infection and reproduction due to its function on asexual reproduction. Rhamnolipids could effectively inhibit growth of hyphae and change cell morphology. Hyphae were blended with each other after treatment with rhamnolipids while in the control they were separated. Shrinkage of protoplast and alteration of the cell membrane and cell wall was also observed through TEM when A. alternata was cultured in a medium amended with rhamnolipids. The reason might be that the structure of the phospholipid bilayer of cell membrane was destroyed by the rhamnolipids acting as same as other surfactants. In this case, the abilities of reproduction and infection of pathogens was greatly reduced, undoubtedly. Rhamnolipids as kinds of biosurfactants have a property of amphiprotic and they generally act on an interface to reduce
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www.soci.org the surface tension between two liquid phases.30 Rhamnolipids obviously affected the cell permeability of hyphae of A. alternata so that the conductivity of supernatants increased and a large amount of DNA and protein leaked from cytoplasm. Although rhamnolipids are regarded as a type of surface active agent, they can not form a uniform and stable system because they are only slightly soluble in water. In order to make rhamnolipids conducive to commercial application, choosing some appropriate solvents to dissolve rhamnolipids to a uniform system is very important.
CONCLUSION The results from this study indicated that rhamnolipids had directly antifungal activities by inhibiting spore germination and mycelium growth of A. alternata. Rhamnolipids decreased fungicidal concentrations of laurel oil because of their damage to cell membrane of A. alternata. However, further experiments will need to be conducted to understand the interaction of rhamnolipids, A. alternata and cherry tomato.
ACKNOWLEDGEMENTS This research was supported by the PhD Programs Foundation of the Ministry of Education of China (20100101110087), the Special Fund for Agro-scientific Research in the Public Interest (200903044), the National Natural Science Foundation of China (31271962 and 30972051), and the Program for Key Innovative Research Teams of Zhejiang Province (2009R50036).
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J Sci Food Agric (2014)
Revised: 21 July 2014
Accepted article published: 28 July 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6845
Biocontrol of post-harvest Alternaria alternata decay of cherry tomatoes with rhamnolipids and possible mechanisms of action Fujie Yan,a,b,c Shixiang Xu,a,b,c Jun Guo,a,b,c Qianru Chen,a,b,c Qin Mengd and Xiaodong Zhenga,b,c* Abstract BACKGROUND: Rhamnolipids were reported to have evident antifungal activity. The efficacy of rhamnolipids against Alternaria alternata and their possible mechanisms involved were investigated. RESULT: The decay incidences of A. alternata of cherry tomatoes (Lycopersicon esculentum) treated by rhamnolipids were significantly reduced. The in vitro assays showed that rhamnolipids inhibited fungal growth on solid medium and prevented spore germination and mycelium growth in liquid medium. In addition, the combination of rhamnolipids and essential oil had a synergistic effect leading to the decrease of fungicidal concentrations of laurel oil. Scanning electron microscopy and transmission electron microscopy observations of the pathogen revealed significant morphological and cell structural alterations in the hyphae. Compared to the control, the content of nucleic acid in supernatant of the suspension of A. alternata increased, while the content of DNA and protein of mycelium decreased, which was in agreement with electrolyte leakage experiments. CONCLUSION: Rhamnolipids could be an alternative to chemicals for controlling post-harvest phytopathogenic fungi on fruits and vegetables. © 2014 Society of Chemical Industry Keywords: rhamnolipids; Alternaria alternata; cherry tomatoes; biological control; mechanisms
INTRODUCTION Considerable post-harvest losses of fruits and vegetables are brought about by decay caused by fungal plant pathogens. Chemical synthetic fungicides are the primary means to control post-harvest diseases but their side-effects such as high residues, long degradation period, strong and acute toxicity, environmental problem and production of fungicide-resistance1,2 make people pay attention to their usage. It is quite apparent that effective, safe and eco-friendly strategies need to be developed for reducing post-harvest losses as well as to make up the shortages of synthetic fungicides. Biosurfactants produced by a variety of microorganisms show unique properties such as mild production conditions and antimicrobial activity,3,4 compared to their chemical counterparts.5,6 Rhamnolipids are formed by one or two rhamnose molecules linked to one or two fatty acids of saturated or unsaturated alkyl chain between C8 and C12 .7 They produced by the bacterium Pseudomonas aeruginosa and are known as efficient anionic biosurfactant molecules.8 They are also used for a wide range of industrial applications, especially in food, cosmetics and pharmaceutical formulations as well as in bioremediation of pollutants.9,10 Rhamnolipids are applicable to a variety of pathogenic microorganisms especially for Gram-positive bacteria,11 – 13 but it is difficult to correlate the bacteriotoxic activity to the properties of rhamnose or fatty acid. It seems that the antimicrobial J Sci Food Agric (2014)
effects result from inhibition of pathogens metabolism, damage of plasma membranes and induction of fruit resistance such as relevant gene expression improvement.14,15 Furthermore, a cell-free culture broth as a crude rhamnolipid product showed the same evident antifungal activity against plant pathogens compared to di-rhamnolipid and mono-rhamnolipid with high purity, which means this crude fermentation product could serve as a potential cost-effective and eco-friendly alternative to chemicals for control of post-harvest phytopathogenic fungi.16 However, using rhamnolipids to control post-harvest diseases of fruits and vegetables also lacks systematic research, especially
∗
Correspondence to: Xiaodong Zheng, Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People’s Republic of China. E-mail: [email protected]
a Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People’s Republic of China b Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, People’s Republic of China c Zhejiang Key laboratory for agro-food processing, Zhejiang University, Hangzhou 310058, People’s Republic of China d Department of Chemical Engineering and Biochemical Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China
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www.soci.org with regard to fungal infections and some possible antifungal mechanisms. The objectives of this study were to evaluate the inhibitory ability of rhamnolipids in vitro and in vivo against A. alternata, a saprophytic pathogen of cherry tomato,17 and to undertake a preliminarily probe into the antifungal mechanisms of rhamnolipids.
MATERIALS AND METHODS Rhamnolipids and essential oil Rhamnolipids obtained from Professor Meng (Chemical Technology Department, Zhejiang University, Hangzhou, China) were produced via fermentation of P. aeruginosa ZJU-211 and purified through ultra-filtration, concentration and separation. The substrates consisted of two types of rhamnolipids: mono- and di-rhamnolipids. Rhamnolipids solutions (100 mg mL−1 ) were prepared with sterile distilled water and diluted to certain concentrations before being used in experiments. Pure-grade essential oil of Laurus nobilis was produced from International Flavors and Fragrances Inc., Shanghai, China. The essential oil was stored in bottles at 4 ∘ C. Fungi and cultures A. alternata was obtained from IMCAS (Institute of Microbiology, Chinese Academy of Science, China) and maintained on the medium of the potato dextrose agar (PDA: extract of 200 mL of boiled potatoes, 20 g of dextrose and 800 mL of distilled water) at 28 ∘ C. Spore suspensions were prepared by flooding a 7-day-old sporulating culture of A. alternata with sterile distilled water and the spore concentration was counted with a haemocytometer.18 In vitro antifungal assay PDA was autoclaved and cooled in a water bath to 40 ∘ C. Rhamnolipids were mixed with sterile molten PDA to obtain the final concentrations of 0, 125, 250, 500 and 1000 μg mL−1 and media were poured into 90-mm Petri plates (15 mL per plate). A 6 mm plug cut from a 7-day-old culture of A. alternata growing on PDA was placed on the centre of each plate and then incubated at 28 ∘ C. Radial growth was measured after 2 days until control group had grown all over the plate. Inhibition (%) = (1 − treatment plaque radius/control plaque radius) × 100. Growth rate (mm day−1 ) = plaque radius of dayn − plaque radius of dayn−1 (n = 3, 4, 5, 6). Five replicates were used for each treatment and experiments were performed twice. Alternaria alternata growth and spore germination assays A spore suspension (100 μL; 105 spores mL−1 ) was dispensed in the flasks containing rhamnolipids at concentrations of 0, 1000 or 2000 μg mL−1 . Dry weight of fungus was measured after a 14 day incubation and results were average of three independent conical flasks for each treatment. For germination assays, rhamnolipids were added to a 10 mL glass tube containing 5 mL PDB to obtain the medium with their final concentrations of 0, 200, 400, 600, 800 and 1000 μg mL−1 respectively. Then a spore suspension (100 μL; 106 spores mL−1 ) was added to each tube. After 20 h of incubation at 28 ∘ C on a rotary shaker (200 × g), at least 100 spores per replicate were observed microscopically to determine germination rate.19 There were three replicates per treatment and the experiment was conducted twice.
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Efficacy of rhamnolipids on inhibition of Alternaria rot in wounded cherry tomatoes Cherry tomatoes without physical injuries and infections were randomly selected. The fruits were gently wounded (6 mm diameter and approximately 3 mm deep) using a sterile borer. Each wound was treated with a solution at 30 μL of rhamnolipids at the concentrations of 0, 500, 1000, 1500 and 2000 μg mL−1 , respectively. After 2 h, 10 μL of spore suspensions of A. alternata (104 spores mL−1 ) were inoculated into each wound. Then the cherry tomatoes were stored in enclosed trays to maintain a high relative humidity at 25 ∘ C. Disease incidence was observed daily after 2 days. There were three replicates per treatment with 20 fruit per replicate and the experiment was conducted twice. Efficacy of rhamnolipids with essential oil from Laurus nobilis in reducing Alternaria alternata Minimal inhibitory concentration and minimal fungicidal concentration Laurel oil was added to a 10 mL glass tube containing 5 mL PDB to obtain the final concentrations ranged from 62.5 to 2000 μg mL−1 with or without rhamnolipids at 500 μg mL−1 . Then a spore suspension of A. alternata (100 μL; 106 spores mL−1 ) was added to each tube for 24 h at 27 ∘ C on a rotary shaker (200 × g). After incubation period, MIC, defined as the lowest concentration of laurel oil that inhibits development of visible microbial growth on PDB was determined. To determine the MFC, subcultures were grown on inhibitor-free nutrient agar plates from each clear tube in the MIC test series, followed by incubation at 27 ∘ C for 48 h.20 In vivo assays Cherry tomatoes were treated as described above. Each wound was treated with 30 μL of (1) sterile distilled water as the control; (2) a solution of rhamnolipids at 500 μg mL−1 ; (3) a solution of laurel oil at 500 μg mL−1 ; and (4) a solution of laurel oil at 500 μg mL−1 with rhamnolipids at 500 μg mL−1 . After 2 h, 10 μL of spore suspensions of A. alternata (104 spores mL−1 ) were inoculated into each wound. Then the cherry tomatoes were stored in enclosed trays to maintain a high relative humidity at 25 ∘ C. Disease incidence was observed daily after 2 days. There were three replicates per treatment with 20 fruit per replicate and the experiment was conducted twice. Efficacy of rhamnolipids in cell permeability of Alternaria alternata Firstly, a 14-day culture of A. alternata in PDB with or without rhamnolipids at 27 ∘ C was centrifuged for the next three experiments. Electrolyte leakage After washing cultures of A. alternata with deionised water, 1 g of sample was transferred into a small beaker and immersed in 20 mL deionised water. Each treatment was divided into two groups. One was exhausted and deflated repeatedly in the vacuum drying oven at 30 ∘ C for 3 h. The other was heated in the boiling water bath for 30 min to make plasma membrane completely destroyed. The conductivity of both specimens was measured using a conductivity meter, and the electrolyte leakage was calculated as the ratio of the conductivities of both groups. Determination of A260 values Through the detection of absorbance at 260 nm, one could estimate the amount of DNA and RNA released from the cytoplasm.
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Biocontrol of A. alternata in cherry tomatoes
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Scanning electron microscopy and transmission electron microscopy A spore suspension (100 μL; 105 spores mL−1 ) was dispensed in the flasks with or without rhamnolipids (1000 μg mL−1 ). After 14 days of incubation at 27 ∘ C on a rotary shaker (200 × g), mycelia were excised from a conical flask for electron microscope sample preparation. Samples were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) overnight at 4 ∘ C, then fixed with 1% osmium tetroxide in phosphate buffer (pH 7.0) for 1 h and rinsed three times in phosphate buffer (pH 7.0). Each specimen was dehydrated by the ethanol solutions with concentrations of 50%, 70%, 80%, 90%, 95%, 100% in turn for 15 min respectively. For scanning electron microscopy (SEM), the specimens were placed in an ethanol and isoamyl acetate mixture for 30 min and transferred to absolute isoamyl acetate for 2 h. The processed samples were observed by SEM.23 For transmission electron microscopy (TEM), the specimens were placed in an absolute acetone for 20 min, in a 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 h, in a 1:3 mixture of absolute acetone and the final Spurr resin mixture for 3 h and in final Spurr resin mixture overnight. After being placed in capsules containing embedding medium and heated at 70 ∘ C for about 9 h, the specimen sections were stained by uranyl acetate and alkaline lead citrate for 15 min, respectively, and observed in TEM.24 Statistical analyses Statistical analyses of the data were performed with SPSS for Windows Version 11.5. One-way ANOVA analyses and Duncan’s multiple range test were used to detect statistical significance, and differences were considered significant when P < 0.05.
50
Inhibition (%)
Measurement of DNA and protein content Total DNA was extracted from 150 mg dry mycelium using the Cetyltrimethyl Ammonium Bromide (CTAB) method. For protein measurement, 150 mg of dry mycelium was ground in liquid nitrogen with a mortar and pestle and transferred to a centrifuge tube containing 2 mL pre-cooled sodium phosphate buffer (pH 7.8). The homogenates were centrifuged at 4 ∘ C at 4000 × g, and the supernatants were assayed for measuring protein content according to Bradford’s method.22 There were three replicates for each treatment and each experiment was repeated twice.
A 60 a
a
a
40
b
30 20 10
c
0
0
125 250 500 -1 Rhamnolipids (µg mL ) control
B Growth rate (mm/day)
The method was according to Hou et al.21 with modifications. The supernatants after centrifugation were read at 260 nm with a nucleic acid analyser.
125
250
1000
500
1000
7 a
6 5
a
b a
b ab
b b b
4 3
a
a a
bc bc
c c
c
b b b
2 1 0
3d
4d
5d
6d
Days
Figure 1. The inhibitory effects of rhamnolipids. (A) Inhibition ratios measured at 6 days. (B) The growth rate of A. alternata in different concentrations of rhamnolipids. Bars represent standard errors of five replicates. Different letter indicates significant differences (P < 0.05) according to the Duncan’s multiple range test.
RESULTS In vitro antifungal assay At the concentration of 125 μg mL−1 , rhamnolipids had a relatively low inhibitory effect on radial growth of A. alternata, with the inhibitory ratio of 26.64%, while increasing to 40.19% at the concentration of 250 μg mL−1 . However, the inhibitory effect was not continuously enhanced when the concentration was higher than 250 μg mL−1 (Fig. 1A). Interestingly, the growth rate significantly slowed down after treated with rhamnolipids at 250 μg mL−1 or above (Fig. 1B), indicating that relative high concentrations of rhamnolipids were effective for delaying fungal growth.
Alternaria alternata growth and spore germination assays Compared to the control, the weight of hyphae was significantly reduced for the treatments at 1000 and 2000 μg mL−1 (Table 1). Spore germination in PDB was strongly inhibited by rhamnolipids and inhibitory ratio increased as the concentration increased. In the control group, 80.6% spores germed and the germ tubes were long and entwined. However, the germination of 64% of spores was inhibited after treated with 400 μg mL−1 and even the quantity was up to 81.7% with rhamnolipids at 1000 μg mL−1 (Fig. 2A).
Table 1. Effect of rhamnolipids (RLs) at concentrations of 0, 1000, 2000 μg mL−1 on A. alternata mycelium in potato dextrose broth medium Concentration of RLs (μg mL−1 ) 0 1000 2000
Dry weight (mg) 409.0 ± 13.53a 108.3 ± 45.37b 97.7 ± 56.58b
Electrolyte leakage (%) 24.2 ± 5.38b 35.9 ± 2.79ab 41.3 ± 0.10a
Protein content of mycelium (mg g−1 ) 13.4 ± 2.80a 13.0 ± 3.68a 4.9 ± 3.31b
DNA content of mycelium (μg g−1 )
A260 value of supernatant
1423.5 ± 332.05a 637.5 ± 69.58b 627.4 ± 98.05b
0.95 ± 0.03b 1.22 ± 0.09b 2.45 ± 0.21a
Values are the mean of three replicates ± SE. Data followed by different letters are significantly different according to Duncan’s multiple range test (P < 0.05).
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Table 2. Antimicrobial activity against A. alternata of laurel oil alone and in the presence of rhamnolipids (RLs) (500 μg mL−1 ) Laurel oil (μg mL−1 )
Parameter MIC MFC
Laurel oil + RLs (μg mL−1 )
1000 1200
500 1000
Disease incidence (%)
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F Yan et al. 100
a
80
Spore germination (%)
a
80 70 60 50 40 30 20 10 0
c
40 20 0
Control
c
e
200
400
600
800
1000
-1
Disease incidence (%)
48h 100
m
60
60h
72h
m
x
80
x
xy
a
m
m
m
b
y
40
c
z cd
20
d
0 0
ELs
RLs+ELs
Treatments
of A. alternata treated with rhamnolipids (Table 1). All these experiments showed that cell permeability of A. alternata obviously increased after treating with high concentrations of rhamnolipids.
cd
d
Rhamnolipids (µg mL )
B
RLs
Figure 3. Effect of laurel oil (500 μg mL−1 ) with rhamnolipids (500 μg mL−1 ) on reduction of A. alternata in cherry tomato fruit. Bars represent standard errors of three replicates. Different letter indicates significant differences (P < 0.05) according to the Duncan’s multiple range test.
b
0
bc
60
MIC, minimal inhibitory concentration; MFC, minimal fungicidal concentration.
A 90
ab
500
1000
1500
2000
-1
Rhamnolipids (µg mL )
Figure 2. (A) Effect of different concentrations of rhamnolipids on spore germination of A. alternata. (B) Effect of rhamnolipids on reduction of A. alternata in cherry tomato fruit. Bars represent standard errors of three replicates. Different letter indicates significant differences (P < 0.05) according to the Duncan’s multiple range test.
In vivo antifungal assay The results showed that disease incidence decreased as the concentration of rhamnolipids increased (Fig. 2B). The percentage of infected fruits was reduced from 70% (control) to 17% (rhamnolipids at 2000 μg mL−1 ) after 60 h of storage. Antimicrobial activity of essential oil alone and in the presence of rhamnolipids Values of minimal inhibitory concentration and minimal fungicidal concentration According to the experimental data, both values of MIC and MFC represented in Table 2 were considerably lower in the presence of rhamnolipids compared with the control. In vivo assays Rhamnolipids or laurel oil at 500 μg mL−1 alone were not effective in inhibiting the A. alternata infections in cherry tomato fruit wounds. However, disease was significantly inhibited with combined treatment, which was better than the treatment with rhamnolipids or laurel oil alone. After 4 days of incubation, the combination treatment reduced the A. alternata incidence to 43% (Fig. 3). Efficacy of rhamnolipids on cell permeability of Alternaria alternata In comparison with the control, electrolyte leakage and values of A260 of the supernatants increased by 71% and 158%, while DNA and protein content in mycelium decreased, after the suspension
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Scanning electron microscopy and transmission electron microscopy Through observation of SEM, A. alternata with no treatment showed even thickness hyphae with normal shapes (Fig. 4A), while the rhamnolipids-treated hyphae exhibited bumpy surfaces and breakage (Fig. 4B). Through observation of TEM, cells with no treatment showed smooth and intact membranes, homogeneous cytoplasms and structured cell walls (Fig. 5A and C). However, after being treated with rhamnolipids at 1000 μg mL−1 , cells exhibited membrane distortion and unclear cell wall structures as well as much smaller size (Fig. 5B and D).
DISCUSSION Rhamnolipids have an evident inhibitory effect on A. alternata, a type of main pathogen that will cause post-harvest diseases of cherry tomato. As the concentrations increased, the fungicidal effect became more remarkable within a certain period of time. However, when concentrations were higher than 3000 μg mL−1 (data not shown), the effect declined instead of being enhanced, and cherry tomato peel was wrinkled and shrank. The possible reason is that the fruit’s ability to resist fungal infection declines because of cell injury when treated with high concentrations of rhamnolipids. Therefore, it is important to choose a suitable concentration of rhamnolipids to apply to post-harvest preservation to make sure that fruit quality is maintained and there is an inhibitory effect. Additionally, as a kind of mild fungistat, rhamnolipids could only inhibit the growth of fungus rather than kill it, which could explain why all treatments had no significant difference on the in vivo assays after 72 h of incubation compared with the control. Undoubtedly, it is necessary to combine rhamnolipids with other safe material with much better effect of fungal inhibition, which can make up the defect that rhamnolipids alone cannot be used for fruit preservation for a long time. Essential oils extracted from plants and natural substances showed an excellent ability to control post-harvest pathogens and diseases in previous studies,23,25 but their inhibitory ability was not stable. They also caused severe fruit injury and a decline in disease resistance if the inhibitory concentration was a little higher. During the present work, we have found that rhamnolipids, as a type of microbial fermentation product, have a mildly inhibitory effect, but when they were combined with laurel oil we have observed a decrease of fungicidal concentration and a better effect on reduction of A. alternata in cherry tomato than the laurel oil alone.
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Biocontrol of A. alternata in cherry tomatoes
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A
B
Figure 4. Scanning electron microscopy photomicrographs of (A) the untreated mycelia of A. alternata (magnification, ×3000); (B) treated with 1000 μg mL−1 rhamnolipids (magnification, ×3000).
A
B
C
D
Figure 5. Transmission electron microscopy photomicrographs of (A) the untreated cell (magnification, ×30 000); (B) treated with 1000 μg mL−1 rhamnolipids (magnification, ×30 000); (C) the untreated cell (magnification, ×50 000); (D) treated with 1000 μg mL−1 rhamnolipids (magnification, ×50 000).
Mechanisms involved in the rhamnolipids inhibition of pathogenic bacteria and fungi have been mainly associated with damage of the microbial cell membrane, reduction of the spore movement ability and spore collapse.26 Moreover, a combination of rhamnolipids and thiosulfonic esters showed a synergisitic effect leading to a decrease of bacterial and fungicidal concentrations of ethyltiosulfonate (ETS) and methyltiosulfonate (MTS). Sotirova et al.20 found rhamnolipids would change the lipid composition of B. subtilis membrane as a result of increasing the content of cardiolipin, a kind of negatively charged phospholipid, and leading to enhancing membrane susceptibility to thiosulfonic esters. Results of our experiments regarding the combination of rhamnolipids and essential oil were similar to their research. We suppose that an interaction of rhamnolipids and plasma membrane of A. alternata allows essential oils to penetrate into cytoplasm more easily so they can exert a greater inhibitory effect. Rhamnolipids have a great inhibitory effect on breeding and growth of fungi by inhibiting their spore germination. A large quantity of hyphae and black spores could be observed in the control group, while the liquid culture with rhamnolipids turned J Sci Food Agric (2014)
to light yellow or even white. Formation and extension of germ tubes played a pivotal role in the pathogenicity of pathogen fungi27 – 29 and lengths of germ tubes were effectively suppressed by high concentrations of rhamnolipids. Obviously, the results of our experiment were consistent with the results reported by Kim et al.26 Mycelium plays an important role in pathogen infection and reproduction due to its function on asexual reproduction. Rhamnolipids could effectively inhibit growth of hyphae and change cell morphology. Hyphae were blended with each other after treatment with rhamnolipids while in the control they were separated. Shrinkage of protoplast and alteration of the cell membrane and cell wall was also observed through TEM when A. alternata was cultured in a medium amended with rhamnolipids. The reason might be that the structure of the phospholipid bilayer of cell membrane was destroyed by the rhamnolipids acting as same as other surfactants. In this case, the abilities of reproduction and infection of pathogens was greatly reduced, undoubtedly. Rhamnolipids as kinds of biosurfactants have a property of amphiprotic and they generally act on an interface to reduce
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www.soci.org the surface tension between two liquid phases.30 Rhamnolipids obviously affected the cell permeability of hyphae of A. alternata so that the conductivity of supernatants increased and a large amount of DNA and protein leaked from cytoplasm. Although rhamnolipids are regarded as a type of surface active agent, they can not form a uniform and stable system because they are only slightly soluble in water. In order to make rhamnolipids conducive to commercial application, choosing some appropriate solvents to dissolve rhamnolipids to a uniform system is very important.
CONCLUSION The results from this study indicated that rhamnolipids had directly antifungal activities by inhibiting spore germination and mycelium growth of A. alternata. Rhamnolipids decreased fungicidal concentrations of laurel oil because of their damage to cell membrane of A. alternata. However, further experiments will need to be conducted to understand the interaction of rhamnolipids, A. alternata and cherry tomato.
ACKNOWLEDGEMENTS This research was supported by the PhD Programs Foundation of the Ministry of Education of China (20100101110087), the Special Fund for Agro-scientific Research in the Public Interest (200903044), the National Natural Science Foundation of China (31271962 and 30972051), and the Program for Key Innovative Research Teams of Zhejiang Province (2009R50036).
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