Curcumin Induces Oxidative Stress in Botrytis cinerea, Resulting in a

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Food Safety and Toxicology

Curcumin induces oxidative stress in Botrytis cinerea resulting in a reduction in gray mold decay in kiwifruit Chenyan Hua, Kai Kai, Wanlin Bi, Wei Shi, Yongsheng Liu, and Danfeng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00539 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Journal of Agricultural and Food Chemistry

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Curcumin induces oxidative stress in Botrytis cinerea resulting in a reduction in

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gray mold decay in kiwifruit

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Chenyan Hua*, Kai Kai*, Wanling Bi, Wei Shi, Yongsheng Liu, Danfeng Zhang#

4 5 6

School of Food and Biological Engineering, Hefei University of Technology, Hefei,

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Anhui 230009, China

8 9 10 11 12

* These authors contributed equally to this work.

13 14

# Corresponding author.

15

Tel:

+86-18755104016.

E-mail:

ACS Paragon Plus Environment

[email protected]


16

ABSTRACT ： Curcumin exhibits an efficient anti-microbial activity; nevertheless,

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its effects on the postharvest decay of fruit have not been examined. Here, effects of

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curcumin on the fruit gray mold of kiwifruit infected by Botrytis cinerea were

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analyzed. Results demonstrated that curcumin induced ROS production and triggered

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apoptosis in B. cinerea hyphae. Use of N-acetyl-cysteine, a ROS scavenger, partially

21

ameliorated the inhibition of curcumin on B. cinerea. The NADPH oxidase inhibitor,

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diphenyleneiodonium chlorine, abrogated the ROS production induced by curcumin,

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suggesting that curcumin induces oxidative stress in B. cinerea via a NADPH oxidase

24

dependent mechanism. Disease severity of gray mold in curcumin-treated kiwifruit

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was significantly reduced. MDA content decreased while antioxidant enzyme activity

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increased in kiwifruit with the application of increasing concentrations of curcumin.

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Collectively, these results indicate that curcumin can be used to control gray mold and

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elevate antioxidant activity in kiwifruit.

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KEYWORDS: curcumin, Botrytis cinerea, ROS, anti-oxidation, kiwifruit


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INTRODUCTION

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Curcumin, a natural polyphenolic compound obtained from Curcuma longa, is

32

commonly used as a spice and food coloring in Asian cooking1. Curcumin has also

33

been considered a promising therapeutic agent that is of clinical interest due to its

34

antioxidant2-4, anti-inflammatory2,5, antimicrobial6,7, and anticancer8-10 properties.

35

Curcumin has been demonstrated to exhibit antifungal activity against the potential

36

human pathogens, including Candida albicans, Sporothrix schenckii, Cryptococcus

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neoformans, Paracoccidioides brasiliensis, and Aspergillus fumigatus6. Several

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phytopathogens, such as Alternaria alternata, Fusarium graminearum, and Botrytis

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cinerea, are also sensitive to curcumin11,12. These latter findings suggest that curcumin

40

has the potential to be used to manage fungal diseases in agricultural crops. Little

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information has been reported, however, on the ability of curcumin to be used to

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manage postharvest diseases of fruits.

43

It has been reported that curcumin exhibits the antifungal activity via targeting

44

multiple signaling molecules at the cellular level. Curcumin induced cell-wall damage

45

and cell death in C. albicans13. Cell membranes were reported to be disrupted in C.

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albicans, resulting in potassium leakage from the cell14,15. Curcumin was also shown

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to act as a pro-oxidant and inhibited the growth of C. albicans by promoting the level

48

of reactive oxygen species (ROS) over a manageable threshold16. Other studies,

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however, have reported that curcumin acts as an antioxidant agent2-4. The action mode

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of curcumin on B. cinerea and whether curcumin has potential to be used to manage

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postharvest decay in fruit crops has yet to be determined. Kiwifruit has attractive taste



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and particularly high content of vitamin C; however, it is very prone to decay during

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storage. B. cinerea is one of the major postharvest pathogens in kiwifruit17. Gray mold

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decay, caused by B. cinerea, can be managed by several synthetic fungicides,

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including flusilazole, fludioxonil, iprodione, tebuconazole, and boscalid18,19. However,

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concerns regarding fungal resistance, environmental contamination, and human health

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issues have made searching for alternatives to synthetic chemical fungicides an active

58

field of research20,21. Therefore, the development and use of natural plant extracts has

59

been explored as an alternative option for postharvest disease control.

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We hypothesize that curcumin can be used as a natural chemical agent for the

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controlling of kiwifruit gray mold. In this study, inhibitory effects as well as the

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possible mechanism of curcumin on kiwifruit gray mold, were explored and

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identified.

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MATERIALS AND METHODS

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Fruit, curcumin, and pathogen. Kiwifruits (Actinidia chinensis cv. Hongyang)

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of uniform size and without any evidence of physical damage were harvested 120 d

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after flowering from an experiment station in Hefei University (Hefei, China) and

69

immediately transported to the laboratory.

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Curcumin (98%) was purchased from Sigma-Aldrich (St. Louis, USA). The

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stock solution (50 g L-1 in DMSO) was filtered through a 0.22 μm filter for

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sterilization and stored at -20 oC for use. The working concentration of curcumin in

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this study was 0, 200, 400, or 600 mg L-1.


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The pathogen B. cinerea FJH5, which was obtained from the Seed Health Centre,

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China Agricultural University, was grown on potato dextrose agar (PDA) at 28 ºC for

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two weeks. Spores were collected and suspended in sterile distilled water. The

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suspension was filtered through two layers of sterile lens cleaning tissue to remove the

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mycelia.

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Inhibitory effect of curcumin on B. cinerea. The MTT test was applied to

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determine the fungal cell viability affected by curcumin according to Patel et al. with

81

slight modification22. Briefly, a 100 μL of B. cinerea spore suspension (1×106 mL-1)

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was inoculated into 5 mL potato dextrose broth (PDB) medium and cultured at 28 ºC

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for 4 d with shaking (200 rpm). Mycelia were partitioned into 100–300 μm pieces,

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followed by treated with curcumin at working concentration for 24 h. Mycelia were

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then harvested and the wet weight was determined. DMSO was applied to remove the

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extra curcumin and then PBS buffer (1 M, pH 7.0) was used to wash the mycelial

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fragments twice. Mycelia were resuspended in 900 μL of PBS and 100 μL of 5 g L-1

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MTT solution. Samples were incubated at 30 ºC in the dark, with shaking for 90 min.

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Mycelial pellet was collected by centrifuge and the MTT-formazan crystallization was

90

resolved by the addition of 800 μL DMSO. The absorbance was determined at 570

91

nm.

92

To test the inhibition of curcumin on mycelial growth of B. cinerea, a 5-mm

93

mycelial disk was taken from a 7-d-old culture and placed in the center of a 90-mm

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Petri dish containing 25 mL of PDA amended with curcumin at each working

95

concentration. Cultures were incubated at 28 ºC and the colony diameters were



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recorded at 3 and 6 d, respectively. PDA plates without curcumin were utilized as the

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control. Three biological replicates were utilized in each treatment and this

98

experiment was repeatedly performed three times.

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Effect of curcumin on hydrogen peroxide (H2O2) accumulation, apoptosis,

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cell death, and potassium release in B. cinerea. A 100 μL of B. cinerea spore

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suspension (1×106 mL-1) was inoculated into 10 mL PDB and cultured at 28 ºC for 4 d

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in a rotary shaker at 200 rpm. Curcumin was then added to the working concentration.

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Mycelia were harvested after 16 and 24 h of treatment to assess H2O2 accumulation

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and cell death. A solution of DMSO was used to wash residual curcumin from the

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mycelia prior to staining for H2O2 to diminish the auto-fluorescence of curcumin that

106

might interfere with the monitoring of the H2O2 signal. Hyphae were stained and

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H2O2 accumulation was evaluated using confocal laser microscopy (LSM710; Zeiss,

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Oberkochen, Germany)23. Fluorescence intensity was analyzed with Image J software.

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Cell apoptosis was analyzed using an Annexin V-FITC/PI kit (BestBio, Shanghai,

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China) according to the manufacturer’s instruction. After treated by curcumin for 24 h,

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hyphae were collected and stained. Control cells were stained for neither annexin

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V-FITC nor propidium iodide. The cells at early stage of apoptosis were positive for

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annexin V-FITC (red fluorescence) but negative for propidium iodide (green

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fluorescence); while those at late apoptotic or necrotic stage would be double stained

115

(yellow, merged with red and green fluorescence)24. Red fluorescence was observed

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using the confocal microscopy at 543 nm excitation and 630 nm emission; and green

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fluorescence was observed at 488 nm excitation and 520 nm emission. Trypan blue


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staining was utilized as described by Lee et al.25 The hyphae stained in dark blue

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indicated a loss of membrane integrity of the cell26. For potassium release assay, 1 ml

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spore suspension was inoculated into 6 mL PDB medium and incubated at 28 ºC in a

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shaker. Mycelia were collected after 4 d of incubation and washed twice using sterile

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distilled water. The washed mycelia were recovered by 6 mL HEPES-NaOH (10 mM,

123

pH 6.5) with 25 mM glucose. Curcumin was added to the working concentrations, and

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Potassium concentration in the suspension was determined by atomic absorption

125

spectroscopy (PerkinElmer AA800, USA)27,28 after 24 h of incubation. Three

126

biological replicates were utilized in each treatment and the experiment was

127

repeatedly performed three times.

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Effect of curcumin on antioxidant enzyme activity in B. cinerea. A 100 μL of

129

B. cinerea spore suspension (1×106 mL-1) was inoculated into 50 mL PDB medium

130

for assessment of the effect of curcumin on antioxidant enzyme activity. Cultures

131

were initially grown at 28 ºC for 7 d in a rotary shaker at 200 rpm. Subsequently,

132

curcumin was added to the working concentrations and mycelia were harvested at 24

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and 48 h after treatment were administered, respectively. Samples of mycelia were

134

immediately frozen and ground in liquid nitrogen. The activities of catalase (CAT)

135

and peroxidase (POD) were measured as the description of Lu et al.29. Protein content

136

was determined by the Bradford assay30. The activities of superoxide dismutase (SOD)

137

and glutathione peroxidase (GSH-PX) were measured using a SOD assay kit

138

(Jiancheng Bioengineering Institute, Nanjing, China) and a GSH-PX assay kit

139

(Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s



140

instructions, respectively. Three biological replicates were utilized in each treatment

141

and this experiment was repeatedly conducted three times.

142

Effect of diphenyleneiodonium chlorine (DPI) and N-acetyl-cysteine (NAC) on

143

the recovery of growth of mycleia treated with curcumin. A 5-mm mycelial disk

144

of B. cinerea obtained from a 7-d-old culture was placed in the center of a 35-mm

145

Petri dish containing 2 mL of PDA amended with curcumin at each working

146

concentration and 0.1 mM of DPI or 10 mM of NAC. Cultures were incubated at 28

147

ºC for 2 d, after which growth of the cultures was assessed. For the detection of

148

hyphae apoptosis, Trypan blue staining was performed as described above. For the

149

determination of H2O2 accumulation in B. cinerea affected by DPI, 0.1 mM of DPI

150

and different working concentrations of curcumin were added to the cultures. Mycelia

151

were collected 24 h after treatment and H2DCFDA staining was performed as

152

described above. Petri dishes containing curcumin without DPI or NAC served as the

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control. Three biological replicates were utilized in each treatment and this

154

experiment was repeatedly conducted twice.

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Development of gray mold on kiwifruit affected by curcumin. Kiwifruit were

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surface disinfected and wounded as the description of Liu et al.31 The curcumin stock

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solution was diluted to the working concentrations with sterile water and 50 μL liquid

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was pipetted to each wound. Fruit were placed in a clean bench until the curcumin

159

solution was absorbed. Five μL of B. cinerea spore suspension (1×104 mL-1) was

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inoculated to each wound. The fruit were arranged in plastic boxes (200 mm × 130

161

mm × 50 mm) containing tap water (not in contact with fruit) in order to maintain the


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relative humidity at about 80% and then stored at 22 ºC in the dark. Lesion diameters

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were recorded after 3, 5, and 7 d of storage, respectively. Three biological replicates

164

were utilized in each treatment and this experiment was repeatedly conducted three

165

times.

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Effect of curcumin on malondialdehyde (MDA) content and antioxidant

167

enzyme activities in kiwifruit. Kiwifruit were immersed in curcumin solution for 1

168

min. Treated fruit were then stored at room temperature in the dark for 48 and 72 h,

169

respectively. Approximately 1 g of tissue was collected, frozen by liquid nitrogen

170

immediately and stored at -20 ºC. The samples were later ground in liquid nitrogen

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prior to determining the MDA content32 and the enzyme activities of CAT, POD,

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SOD, and GSH-PX. Three biological replicates were utilized in each treatment and

173

this experiment was repeatedly conducted three times.

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Expression level of NOXR in B. cinerea. A 100 μL of B. cinerea spore

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suspension (1×106 mL-1) was inoculated into 10 mL of PDB and incubated at 28 ºC

176

for 4 d in a rotary shaker. Curcumin were added to the working concentrations. At 24

177

h after curcumin treatment, mycelia were harvested, frozen in liquid nitrogen, and

178

stored at -80 ºC. Total mRNA was extracted and RT-qPCR was performed according

179

to Zhang et al.33 Gene-specific primers were presented in Table 1. The expression

180

level of NOXR was calculated by the 2−ΔΔCT method34 using Actin as the reference

181

gene. Treatment without curcumin served as the control. Three biological replicates

182

were utilized in each treatment, and the experiment was repeatedly performed twice.

183

Statistical Analysis. The statistical analyses were conducted using SAS 9.2



184

(Cary; NC, USA). Data were analyzed using a one-way ANOVA and shown as mean

185

± standard errors of least square means (SEM). The p-value < 0.05 was accepted as

186

statistically significant according to Duncan’s multiple range tests. .

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RESULTS AND DISCUSSION

189

Curcumin reduced cell viability and retarded vegetative growth of B.

190

cinerea. Curcumin was observed to decrease the viability of human cancer cells,

191

including L929 and MA104 cells35, gallbladder carcinoma cells36, and breast cancer

192

cells37, by MTT assay. However, this effect on fungi, especially on B. cinerea cell,

193

has not been studied previously. As shown in Figure 1A, absorbance of

194

MTT-formazan decreased in the curcumin treatment. In 200 mg L-1 curcumin

195

treatment, absorbance was markedly lower (p < 0.05) than the control, suggesting that

196

the viability of B. cinerea cell was reduced by exposure to curcumin. Correspondingly,

197

the vegetative growth of B. cinerea was notably (p < 0.01) suppressed in each

198

curcumin treatment as well (Figure 1B). At 6 d after inoculation, the colony diameter

199

of the 600 mg L-1 curcumin treatment was reduced by 57.3% compared with the

200

control. This result provides direct evidence that curcumin exhibits a significant

201

inhibitory effect on B. cinerea. However, the action mode of curcumin on B. cinerea

202

still remains largely unclear.

203

Curcumin promotes ROS accumulation in B. cinerea. A significant induction

204

of ROS in B. cinerea hyphae was observed at 16 h after the hyphae were treated with

205

curcumin (Figure 2). Results indicated that ROS accumulation in B. cinerea induced


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by curcumin occurred in a dose dependent manner. At 16 h after treatment, hyphae

207

stained with annexin V–FITC increased with increasing level of curcumin, indicating

208

that curcumin triggered cell apoptosis in B. cinerea. Hyphae co-stained with annexin

209

V–FITC and propidium iodide also increased, suggesting that the number of late

210

apoptosis/dead hyphae increased in response to curcumin (Fig. 3A). This phenomenon

211

was more obvious at 24 h with exposure to curcumin. Trypan blue staining revealed

212

that dark blue staining of hyphae was also promoted in a dose dependent manner by

213

exposure to curcumin (Figure 3B). Potassium concentrations in the samples treated by

214

400 and 600 mg L-1 curcumin were significantly increased (Figure 4), suggesting that

215

cell

216

Curcumin-induced membrane disruption and leakage of potassium ions from the

217

cytosol has been previously reported15. Sharma et al.16 reported that curcumin induced

218

ROS accumulation in C. albicans and inhibited vegetative growth. ROS induced by

219

curcumin had also been observed in mouse fibroblast cells38, cutaneous T-cell

220

lymphoma cultures10, and leukemic cells39. High level of ROS production can induce

221

oxidative stress, resulting in the disruption of cell membranes40-42. Results of the

222

present study indicated that curcumin also induced ROS accumulation in B. cinerea,

223

and a concomitant disruption of cell membranes, in a dose dependent manner.

224

Curcumin was also shown to disrupt fungal cell membrane via other mechanisms,

225

such as the suppression of ergosterol biosynthesis14.

membrane

permeability

was

affected

by

exposure

to

curcumin.

226

Since curcumin stimulated ROS production in B. cinerea, the activities of the

227

antioxidant enzymes, CAT, POD, SOD, and GSH-PX in hyphae were also determined.



228

CAT activity was affected (p < 0.05) by curcumin at 600 mg L-1 at 24 h and induced

229

significantly (p < 0.01) in response to curcumin at 400 or 600 mg L-1 at 48 h (Fig. 5A).

230

The POD activity in the fungal hyphae was also induced (p < 0.05) by ≥ 400 mg L-1

231

curcumin (Fig. 5B). The activity of SOD was elevated but not significantly greater

232

than the control until the curcumin concentration was as high as 600 mg L-1 (Fig. 5C).

233

At 24 h after treatment, GSH-PX activity in hyphae in each curcumin treatment was

234

markedly (p < 0.01) higher than the control. A difference in GSH-PX activity was

235

also observed at 48 h in response to 600 mg L-1 curcumin (Fig. 5D). Previous studies

236

reported that these antioxidant enzymes are induced by H2O2 and reflect H2O2

237

accumulation43,44. Our present data on the effect of curcumin supported these previous

238

studies.

239

The mechanism of ROS generation induced by curcumin has not been entirely

240

elucidated. Swatson et al.45 reported that curcumin stimulated ROS production in

241

Dictyostelium discoideum via a protein kinase A (PKA) dependent mechanism. A

242

pull-down assay showed that curcumin targets a series of proteins which play roles in

243

ROS metabolism, including glutathione S-transferase and NAD(P)H dehydrogenase,

244

resulting in increased ROS levels in cells39.

245

In the present study, vegetative growth inhibition could be partially inhibited by

246

the addition of the antioxidant enzyme, NAC, which is in agreement with previous

247

studies on C. albicans16 and human cancer cells46-48. Trypan blue staining also

248

indicated that NAC prevented cell death promoted by curcumin (Fig. 6B), suggesting

249

the pro-oxidative activity of curcumin is partially responsible for its ability to inhibit


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fungal growth. Mitochondrial respiratory chain and NADPH oxidase are the main

251

sources for ROS production within49,50. However, the application of DPI, a NADPH

252

oxidase inhibitor, did not prevent the inhibition on vegetative growth by curcumin in

253

culture (Fig. 6A). Trypan blue staining also revealed that the cell death promoted by

254

curcumin was not ameliorated by DPI (Fig. 6B). Compared with the treatments with

255

only curcumin, we observed that the addition of DPI noticeably decreased ROS

256

accumulation induced by curcumin (Fig. 7A). Moreover, the expression of a subunit

257

of NADPH oxidase, NOXR, was clearly induced by curcumin as well (Fig. 7B). This

258

result implicated that the ROS burst in B. cinerea induced by curcumin was mediated

259

by NADPH oxidase. This premise was in agreement with a previous study in D.

260

discoideum45, where it was reported that PKA plays a role upstream of NADPH

261

oxidase in the ROS burst51. While DPI also inhibited nitric oxide production in cells,

262

it was suggested that the addition of DPI might be cytotoxic to B. cinerea, leading to a

263

more severe inhibition of mycelial growth.

264

Curcumin inhibits gray mold in kiwifruit. Botrytis cinerea is an important

265

phytopathogen and is responsible for up to 25% yield losses in kiwifruit. Previous

266

studies have indicated that curcumin is a potential effective antifungal agent that can

267

be used in clinical treatments of human diseases6. Chen et al.12 reported that Curcuma

268

longa extracts containing curcumin inhibited the growth of B. cinerea effectively and

269

the EC50 was 310 mg L-1. This finding suggests that curcumin may be useful as an

270

alternative to synthetic fungicides for postharvest protection. The photosensitization

271

of curcumin followed by blue light irradiation diminished the fungal contamination of



272

date fruit and effectively extended the shelf life52. Whereas, the ability of curcumin to

273

provide postharvest protection on fruit has not been previously evaluated.

274

Results of the present study indicate lesion diameter in kiwifruit treated at all the

275

concentrations of curcumin decreased (p < 0.05) compared with the control (Fig. 8).

276

At 7 d after treatment, lesion diameters in pathogen-inoculated wounds treated by 200,

277

400, and 600 mg L-1 curcumin was significantly (p < 0.01) smaller by 15, 15, and 20%

278

relative to the control, respectively. This result indicates that the application of

279

curcumin effectively decreased disease severity.

280

Curcumin exhibits antioxidant activity in kiwifruit. The excessive

281

accumulation of ROS leads to oxidative stress and results in injury or cell death53,54.

282

Therefore, we investigated whether curcumin induced ROS accumulation in kiwifruit

283

tissue and accelerated fruit senescence. Results indicated that MDA content, an

284

indicator of cell injury, was notably (p < 0.05) lower in the fruit treated by curcumin

285

than the control (Fig. 9). At 72 h after treatment, the MDA level in fruit treated by 200,

286

400, and 600 mg L-1 curcumin was 18.8, 47.7, and 73.3% lower than in the control,

287

respectively.

288

The antioxidant properties of curcumin were previously studied3,55,56. It showed

289

that curcumin suppressed lipid peroxidation, scavenged superoxide as well as peroxyl

290

radicals effectively57,58. About 97% of the lipid peroxidation in a linoleic acid

291

emulsion was reported to be inhibited by the supplementation of 15 mg L-1 curcumin3.

292

As a natural phenolic compound, curcumin has antioxidant activity directly59.

293

Moreover, it induces antioxidant enzyme activity in cells as well. In the present study,


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the CAT, POD, SOD, and GSH-PX activities in kiwifruit tissue was determined at 24,

295

48, and 72 h after treated with various concentrations of curcumin, respectively (Fig.

296

10). The CAT activity was notably (p < 0.05) higher than the control at 48 and 72 h

297

after treatment; while the POD, SOD, and GSH-PX activities were higher (p < 0.05)

298

compared with the control at each time point. Similar results were observed in

299

experiments utilizing rats and mice60,61.

300

Curcumin has been reported to exhibit both antioxidant and pro-oxidant activities,

301

depending on the concentrations applied62. In that study, curcumin at low levels

302

prevented GSH depletion, but decreased GSH activity at higher levels. Notably,

303

curcumin has the ability to scavenge peroxyl radicals. Therefore, we hypothesize that

304

plant and fungal cells have different levels of sensitivity to curcumin. We further

305

suggest

306

were high enough to induce oxidative stress in fungi but not in kiwifruit. Importantly,

307

no evidence of ROS production, as measured by fluorescent dye staining, was

308

observed in kiwifruit treated with curcumin (data not shown).

that

the

concentrations

of

curcumin

used

in

this

experiment

309

In conclusion, curcumin induced excessive ROS production in B. cinerea,

310

leading to cell apoptosis and vegetative growth retardation in a dose dependent

311

manner. Due to this activity, gray mold infection in wounded-inoculated kiwifruit was

312

also inhibited. Curcumin also exhibited antioxidant activity in kiwifruit. Based on

313

these results, we suggest that curcumin should be considered a novel antifungal agent

314

that can be used for management of postharvest diseases of harvested fruit crops.

315



316

AUTHOR INFORMATION

317

Corresponding Author #

318

Telephone: +86-187-5510-4016. E-mail: [email protected].

319

ORCID

320

Danfeng Zhang: 0000-0002-8776-9829

321

ACKNOWLEDGEMENTS

322

We thank the National Natural Science Foundation of China for financial support

323

(Grant No. 31500214) and Prof. Michael Wisniewski from USDA for manuscript

324

editing.

325

NOTES

326

The authors declare no competing financial interest.

327 328

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514

Table 1

515

Primers used for the RT-qPCR analysis. gene name

accession number

primer sequences (5→ 3′)

product length (bp)

NOXR

BC1G_06200.1

F: TATCAAAAAGCCGTTCGCCT

145

R: CGATCATATTGTTTCCCCTCAAG Actin

XM001553318

F: CTCTATTCAAGCCGTCCTCTCC R: TAATCAGTCAAATCACGACCAGC


162


517

Figure captions

518 519

Figure 1. Effect of curcumin on the cell viability and vegetative growth of B. cinerea.

520

The cell MTT formazan production in B. cinerea was determined at 24 h after

521

treatment (A). Colony diameter was recorded after 3 and 6 d of incubation,

522

respectively (B). Values with asterisks indicate significant differences (*, p < 0.05; **,

523

p < 0.01) from the control according to Duncan’s multiple range test. Data are

524

expressed as mean ± SEM (n = 9).

525 526

Figure 2. H2O2 accumulation and cell death in hyphae of B. cinerea treated by

527

curcumin. Green fluorescence in the hyphae indicates the accumulation of H2O2 (A),

528

and the fluorescence intensity was calculated (B). Values with asterisks indicate

529

significant differences (**, p < 0.01) from the control according to Duncan’s multiple

530

range test. Data are expressed as mean ± SEM (n = 9). Bar = 50 μm.

531 532

Figure 3. Induction of apoptosis by curcumin in B. cinerea. The hyphae stained with

533

annexin V–FITC alone (red fluorescence) were at early apoptosis stage, while those

534

co-stained with annexin V–FITC and propidium iodide (yellow fluorescence) were at

535

late apoptosis stage or dead (A). Cell death was indicated by the accumulation of dark

536

blue staining with Trypan blue (B). Bar = 50 μm.

537 538

Figure 4. Potassium leakage from B. cinerea after 24 h of incubation with curcumin.


Page 26 of 39

Page 27 of 39


539

Values with asterisks indicate significant differences (**, p < 0.01) from the control

540

according to Duncan’s multiple range test. Data are expressed as mean ± SEM (n = 9).

541 542

Figure 5. Antioxidant enzyme activity in hyphae of B. cinerea treated by curcumin.

543

Activities of catalase (CAT, A), peroxidase (POD, B), superoxide dismutase (SOD,

544

C), and glutathione peroxidase (GSH-PX, D) was determined at 24 and 48 h after

545

treatment, respectively. Values with asterisks indicate significant differences (*, p

Curcumin Induces Oxidative Stress in Botrytis cinerea, Resulting in a

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