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In vitro and in vivo effectiveness of phenolic compounds for the control of postharvest gray mold of table grapes
Postharvest Biology and Technology 139 (2018) 106–114
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
In vitro and in vivo effectiveness of phenolic compounds for the control of postharvest gray mold of table grapes
T
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Dandan Xua, Yizhen Dengb, Tingyu Hana, Liqun Jiangb, Pinggen Xib, Qi Wanga, Zide Jiangb, , ⁎ Lingwang Gaoa, a
College of Plant Protection, China Agricultural University, Beijing 100193, China Department of Plant Pathology/Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Antifungal activity Piceatannol Postharvest decay Pterostilbene
As the natural antimicrobial metabolites, phenolic compounds were reported to be effective in the inhibition of phytopathogenic fungi, including the postharvest decay agents. However, comprehensive study on the biological activity of phenolic compounds and their application on controlling postharvest gray mold of table grapes is lacking. In this study, the antifungal effect of 18 natural or synthetic phenolic compounds purchased from commercial suppliers, including simple phenolic, phenolic acids, stilbenes and flavonoids, were determined on four gray mold strains by an in vitro agar dilution assay. Overall, seven phenolic compounds were effective on inhibiting B. cinerea growth and were selected to test their activity on conidial germination as well as in vivo application on grape berries. Pterostilbene showed the highest antifungal activity and greatly reduced the growth of the mycelia, caused hyphae deformation, suppressed conidial germination of B. cinerea, and completely inhibited the germination of conidia at the concentration of 50 mg L−1. Furthermore, treatment of grape berries with pterostilbene and piceatannol significantly reduced the disease incidence and severity. Our results demonstrate the antifungal activity of phenolic compounds and highlight their potentials as an alternative strategy in the control of postharvest gray mold of table grapes.
1. Introduction The fungus Botrytis cinerea causes gray mold, leads to huge postharvest losses and is considered as the main postharvest decay of table grapes (Vitis vinifera) (Romanazzi et al., 2012; Youssef and Roberto, 2014). In many countries, synthetic fungicides are not allowed to use for the control of postharvest decay of table grapes, and only sulfur dioxide (SO2) is permitted to use as an adjuvant. Alternatives to SO2 are urgently required in view of the hazardous effect to human health caused by SO2 residues and the bleaching injuries in berries caused by the application of SO2 (Gándara-Ledezma et al., 2015; Parafati et al., 2015; Romanazzi et al., 2016a). Among numerous unconventional control strategies, the induction of fruit resistance via application of plant or microorganism products with antimicrobial activity and the physical means can be considered, either alone or as a part of an integrated pest management policy (Romanazzi et al., 2016b). Within plant natural products, phenolic compounds, which widely distribute in the tissues of resistant
grapevine cultivars as important secondary metabolites, are thought to be involved in defence response against fungi by forming a chemical barrier that limits pathogen growth and increase plant resistance (Mlikota Gabler et al., 2003; Del Rio et al., 2004; Pizzolitto et al., 2015). The fungus B. cinerea is present in vineyards as part of the environmental microflora, infection of grape often occurs at bloom time, followed by a period of latency, and generally causes disease symptoms when berries begin to ripen and meanwhile phenolic compounds contents changes (Holz et al., 2003; Keller et al., 2003; Liang et al., 2011). Therefore, the phenolic compounds (phytoanticipins) that are constitutively present in grape berries and with potential in inhibiting B. cinerea growth are of great interest. Accumulation of certain phenolics are induced as a response to biotic and abiotic stress, and these metabolites are referred to as phytoalexins (Ahuja et al., 2012). Some phytoalexins have broad spectrum of anti-pathogen activities and their accumulation in plants can promote host defense response, e.g. the accumulation of trans-resveratrol and catechin in grapes exposed to chitosan and UV-C (Romanazzi et al., 2006), and the accumulation of
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Corresponding authors at: College of Plant Protection, China Agricultural University, Beijing 100193, China E-mail addresses: [email protected] (D. Xu), [email protected] (Y. Deng), [email protected] (T. Han), [email protected] (L. Jiang), [email protected] (P. Xi), [email protected] (Q. Wang), [email protected] (Z. Jiang), [email protected] (L. Gao). https://doi.org/10.1016/j.postharvbio.2017.08.019 Received 10 February 2017; Received in revised form 2 August 2017; Accepted 27 August 2017 0925-5214/ © 2017 Published by Elsevier B.V.
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Fig. 1. Structures of the 18 tested phenolic compounds.
biological activity of phenolic compounds against B. cinerea, as well as practical feasibility of their application for protection of table grapes form postharvest gray mold. In this study, 18 phenolic compounds including simple phenolic, phenolic acids, stilbenes, flavonoids that were previously reported to have antimicrobial activity, were selected and evaluated in the aspect of their antifungal activity against B. cinerea, both in vitro and in vivo.
stilbenic phytoalexins (resveratrol and viniferin) in grapevine treated with rhizospheric bacteria (Aziz et al., 2016), associated with the development of resistance against B.cinerea. At present, several phenolic compounds were proved to have antifungal properties against Fusarium graminearum (Gauthier et al., 2016), Alternaria alternata (Wang et al., 2017), Penicillium expansum (Sanzani et al., 2014), Plasmopara viticola (Gabaston et al., 2017) and B. cinerea (Mendoza et al., 2013), making them as good “natural pesticide” candidates for improving plant resistance to phytopathogen. However, little is known about the 107
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compound was determined at 72 h post B. cinerea inoculation. The data were presented with a hierarchical clustering of mycelial growth for each fungus in the presence of 200 mg L−1 phenolic compounds, with the control condition set as 100%, which were organized and visualized using the hierarchical clustering software Gene Cluster 3.0 (De Hoon et al., 2004) and Tree view software (Page, 1996). To assess the absolute effectiveness of some active phenolics on the B. cinerea, the concentration inhibiting 50% of fungal growth (IC50) was measured under various concentrations. Mycelium morphology of tested fungal samples was examined and imaged by optical microscopy (Eclipse Ni, Nikon), equipped with Nikon DS-Ri1 camera. For conidial germination assay, B. cinerea spore suspension was prepared by washing the conidia with PDB (potato dextrose broth medium, 37.5 g potato, 20 g dextrose per liter) and the final concentration was adjusted as 105 conidia per milliliter. Then, equal volume of phenolic compounds or ethanol, as solvent control, was added into the spore suspension (Zheng et al., 2011). All tested samples were kept in dark to protect the phenolics (Adrian and Jeandet, 2012), at 22 °C. Conidial germination was determined by calculating the percentage of germinated spores. Each treatment was repeated three times.
Table 1 Technical facts of compounds used in this study. Phenolic compound
CAS
Catalog number
Source
Resveratrol Luteolin Apigenin Catechol Ferulic acid Catechin Coumarin Caffeic acid Quercetin ρ-coumaric acid Sinapic acid Naringenin Gallic acid Piceid Pterostilbene Kaempferol Protocatechuic acid Piceatannol
501−36-0 491−70-3 520−36-5 120−80-9 537−98-4 154−23-4 91−64-5 331−39-5 117−39-5 501−98-4 530−59-6 480−41-1 149−91-7 65914−17-2 537−42-8 520−18-3 99−50-3 10083−24-6
V900386 79506 M01289 359694 408509 C217500 107521 247526 268623 C9008 397578 393137 210917 296286 455753 258643 223812 P1928
Sigma-Aldrich Fluorochem Ltd Fluorochem Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd
2. Materials and methods
2.5. In vivo bioassays
2.1. Fruit material
The antifungal activity of phenolic compounds against B. cinerea was assessed on grape berries by wounded assay (Sanzani et al., 2009). Two mm deep wounds in the equatorial zone were made by dissected needles and inoculated with 10 μL of phenolic solution (104 mg L−1, 100 μg per wound). After drying at room temperature for approximately 30 min, each wound was inoculated with 10 μL of a 5 × 104 conidia per milliliter suspension of B. cinerea and allowed to air-dry for 1 h. Wounds with equal volume of solvent and inoculated with the B. cinerea severed as a control. The experiment repeated twice with three replicates, each consisting of 45 berries per treatment. All treated berries were sealed in plastic containers and kept at 22 °C in dark and approximately 80% relative humidity for 9 days. Disease symptom was examined by visual evaluation, in which grape berries showing apparent water-soaked lesions with mycelia and sporulation beyond the wounded area were considered as infected (Wang et al., 2015). Disease incidence was calculated as percentage of infected berries and the rot lesion diameters were recorded.
Fresh table grape berries (cv Mare’s milk) were carefully removed from the rachis, keeping pedicels intact. The berries without visible defects or injuries, and with uniform size and ripening stage were chosen. Before treatments, the selected berries were randomized, surface sterilized in a 2% sodium hypochlorite solution for 2 min, rinsed under running tap water for 1 min, and air-dried at room temperature. 2.2. Plant pathogen B. cinerea isolates TGM and BGM used in this study were kindly provided by the MOA Key Laboratory of Plant Pathology (China Agricultural University, Beijing), and B. cinerea isolates GBR and GBW were obtained from table grape berries with typical gray mold symptoms. All fungal strains were maintained on PDA medium (potato dextrose agar medium, 200 g potato, 20 g dextrose and 15 g agar per liter) at 4 °C until use.
2.6. Statistical analysis 2.3. Chemical reagents Data were statistically analyzed using analysis of variance (ANOVA) and expressed as mean ± standard deviation (SD). The mean separations were analyzed using Duncan’s multiple range tests and differences among different treatments was determined at 5% level (IBM SPSS Statistics 22, USA).
All phenolic compounds (Fig. 1) used in the study had a purity grade higher than 98%. Resveratrol was purchased from Sigma-Aldrich (St. Louis, MO, USA), and apigenin and luteolin were purchased from Fluorochem Ltd (Old Glossop, Derbyshire, UK), the other tested phenolic compounds were purchased from J&K Scientific Ltd (Beijing, China). Detailed information of tested compounds listed in Table 1. All compounds were dissolved in ethanol to prepare stock solutions.
3. Results and discussion 3.1. In vitro assessment of anti-fungal activity for 18 phenolics
2.4. In vitro bioassays The 18 tested phenolic compounds differentially affected fungal growth and sensitivity toward phenolics differed among the four tested strains (Table 2). Six compounds (catechol, resveratrol, coumarin, naringenin, pterostilbene and piceatannol) displayed antifungal activity (growth inhibition more than 20%) and protocatechuic acid enhanced the growth for all tested strains. Kaempferol exhibited no activity (growth affected less than 6%) in all tested strains. However, some phenolic compounds showed differential effects on mycelial growth of the four tested fungal strains, i.e. ferulic acid suppressed B. cinerea isolates GBR, GBW and BGM growth, while enhanced the growth of TGM. Ten out of 18 tested molecules had an inhibitory activity on fungal growth for all the four strains tested, one stimulated growth for the four
The antifungal activity of phenolic compounds was assessed according to Lambert et al. (2012). Phenolic compounds were added to the cooled PDA medium respectively, with an equal volume of ethanol added to the PDA medium as solvent control. Plug inocula of 6 mm diameter were cut from the actively growing fungal cultures and then placed at the center of PDA plate, with or without phenolic supplements. The cultures were incubated at 22 °C in the dark. Colony diameter (in mm) was measured at 24-h intervals for five consecutive days. Each phenolic compound was assessed on the four isolates. Four independent replicates were performed for each instance and experiment was carried out twice. The percentage of growth inhibition or induction for each phenolic 108
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Table 2 Effect of phenolic compounds on the colony diameter of B.cinereaTable 2 (Continued). Phenolic compounds
Concentration (mg L−1)
Mycelial growth of different tested strains (mm) GBR
GBW
BGM
TGM
ρ-coumaric acid
0 50 100 200
65.4 44.3 38.8 37.0
± ± ± ±
3.4 1.5 0.8 1.0
64.9 56.5 55.5 53.0
± ± ± ±
2.5 1.3 0.5 1.0
63.6 56.5 54.7 46.1
± ± ± ±
2.4 1.3 1.2 0.1
69.6 54.8 50.9 49.9
± ± ± ±
0.7 0.8 0.8 1.5
Resveratrol
0 50 100 200
63.5 62.5 57.7 51.8
± ± ± ±
1.3 1.5 1.1 0.7
65.3 59.2 58.5 46.2
± ± ± ±
1.1 1.0 2.5 0.4
72.4 67.5 63.3 54.7
± ± ± ±
0.8 1.3 1.5 1.6
71.3 64.3 55.2 44.1
± ± ± ±
0.9 2.1 2.7 1.1
Sinapic acid
0 50 100 200
69.0 63.3 61.8 60.9
± ± ± ±
1.4 1.3 2.0 1.2
64.9 61.8 60.3 58.8
± ± ± ±
2.5 0.4 1.2 0.9
66.3 60.0 59.8 57.3
± ± ± ±
0.4 1.0 1.0 0.4
69.6 60.4 59.3 56.8
± ± ± ±
0.7 0.6 0.8 0.9
Coumarin
0 50 100 200
67.2 66.1 62.8 55.1
± ± ± ±
1.2 1.7 3.9 0.9
65.8 62.8 58.8 53.1
± ± ± ±
1.1 0.6 0.6 0.8
60.8 56.8 54.5 53.6
± ± ± ±
1.1 2.4 3.0 0.4
61.8 57.6 55.8 49.1
± ± ± ±
0.8 0.5 1.8 1.7
Catechol
0 50 100 200
59.4 55.3 49.6 38.2
± ± ± ±
0.8 0.3 1.2 0.7
48.9 45.7 38.3 26.8
± ± ± ±
1.9 3.4 2.3 3.5
57.2 56.0 52.5 40.0
± ± ± ±
1.5 2.2 0.9 0.4
57.9 57.0 44.3 34.9
± ± ± ±
1.7 3.0 3.6 3.3
Piceatannol
0 50 100 200
52.3 46.0 39.5 39.1
± ± ± ±
3.2 0.9 1.3 0.7
64.5 54.5 48.3 38.7
± ± ± ±
0.7 2.6 0.8 2.3
65.7 63.8 54.5 52.3
± ± ± ±
4.2 2.0 2.2 1.8
57.0 52.3 49.8 40.5
± ± ± ±
0.7 1.9 2.9 4.6
Naringenin
0 50 100 200
59.4 57.7 46.8 34.9
± ± ± ±
0.8 2.8 2.3 1.5
48.9 46.9 40.4 30.9
± ± ± ±
1.9 1.2 0.9 0.5
57.2 51.8 43.1 35.8
± ± ± ±
1.5 0.5 2.2 1.2
57.9 42.3 41.4 36.1
± ± ± ±
1.7 2.6 4.1 2.1
Pterostilbene
0 50 100 200
58.5 33.8 31.3 30.3
± ± ± ±
0.9 0.5 0.4 0.3
56.4 35.7 33.8 32.6
± ± ± ±
2.0 0.5 1.3 1.0
66.8 42.3 37.9 35.1
± ± ± ±
1.8 2.4 2.0 2.7
53.2 33.9 31.9 31.8
± ± ± ±
1.3 2.8 1.4 1.8
Kaempferol
0 50 100 200
66.3 63.0 63.3 62.8
± ± ± ±
1.4 1.5 0.7 0.8
61.9 62.3 60.2 57.9
± ± ± ±
1.4 1.0 5.8 4.4
56.7 57.0 54.6 56.4
± ± ± ±
0.6 3.5 1.4 1.1
50.3 48.3 47.0 45.3
± ± ± ±
2.1 2.5 1.7 1.3
Ferulic acid
0 50 100 200
63.5 60.5 55.7 50.0
± ± ± ±
1.3 2.3 1.5 0.9
63.2 59.8 56.9 50.3
± ± ± ±
3.7 1.8 0.8 0.8
82.7 76.0 66.0 58.8
± ± ± ±
2.1 3.5 2.6 0.9
47.0 65.7 58.9 52.7
± ± ± ±
2.8 1.5 1.0 1.5
Caffeic acid
0 50 100 200
67.2 65.2 61.5 58.7
± ± ± ±
1.2 2.5 1.0 2.4
65.8 61.5 60.3 59.1
± ± ± ±
1.1 1.0 2.1 1.0
60.8 59.1 57.5 59.3
± ± ± ±
1.1 1.0 1.5 1.2
61.8 57.7 59.0 60.2
± ± ± ±
0.8 0.8 1.3 2.3
Luteolin
0 50 100 200
66.3 57.8 57.5 57.3
± ± ± ±
1.4 2.2 1.3 0.8
61.9 59.1 58.9 58.8
± ± ± ±
1.4 6.6 2.6 1.4
56.7 57.2 56.5 54.5
± ± ± ±
0.6 1.1 4.8 1.3
50.3 53.3 53.8 50.3
± ± ± ±
2.1 1.9 2.1 1.4
Apigenin
0 50 100 200
66.3 59.6 57.4 56.8
± ± ± ±
1.4 2.3 0.8 4.9
62.3 61.9 59.3 57.7
± ± ± ±
1.4 1.4 1.9 2.5
56.7 61.8 59.4 52.5
± ± ± ±
0.6 2.0 5.6 0.9
53.3 49.0 50.3 50.0
± ± ± ±
1.5 2.6 2.1 0.9
Catechin
0 50 100 200
67.3 63.5 67.7 72.0
± ± ± ±
1.6 1.5 2.4 1.0
72.6 76.9 78.4 76.5
± ± ± ±
0.5 0.5 1.5 1.3
68.7 68.0 71.2 76.8
± ± ± ±
1.2 2.9 0.8 0.7
61.2 54.1 54.4 57.3
± ± ± ±
1.3 1.2 0.8 2.2
Quercetin
0 50 100 200
61.8 59.8 58.2 57.0
± ± ± ±
1.8 0.6 0.7 0.5
59.8 60.2 60.8 61.9
± ± ± ±
2.1 1.3 1.2 0.5
60.7 65.4 67.3 69.4
± ± ± ±
2.3 1.7 2.1 1.2
59.3 60.3 60.9 61.6
± ± ± ±
1.3 1.5 2.3 1.6
Gallic acid
0 50 100 200
59.4 60.7 57.6 56.3
± ± ± ±
0.8 0.6 4.1 4.3
48.9 53.8 55.4 53.7
± ± ± ±
1.9 0.8 1.2 0.3
57.2 61.7 63.7 62.8
± ± ± ±
1.5 5.1 3.3 1.4
57.9 61.7 60.2 61.7
± ± ± ±
1.7 3.3 0.6 3.4
(continued on next page)
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Table 2 (continued) Phenolic compounds
Concentration (mg L−1)
Mycelial growth of different tested strains (mm) GBR
GBW
BGM
TGM
Piceid
0 50 100 200
58.5 60.6 61.3 57.9
± ± ± ±
0.9 1.0 1.0 3.8
56.4 56.3 59.8 60.1
± ± ± ±
2.0 0.8 2.0 1.3
66.8 64.7 68.3 71.1
± ± ± ±
1.8 4.5 1.9 2.0
53.2 56.4 57.1 57.3
± ± ± ±
1.3 4.3 2.1 0.9
Protocatechuic acid
0 50 100 200
50.8 55.6 58.0 58.5
± ± ± ±
2.0 1.0 0.9 2.0
26.2 29.3 32.0 32.8
± ± ± ±
0.3 1.6 0.5 2.1
63.8 62.0 64.3 65.8
± ± ± ±
1.4 0.5 0.7 0.3
51.7 58.0 59.3 61.3
± ± ± ±
1.2 0.9 3.2 0.9
Each value is the mean colony diameter ( ± SD) after 72 h of incubation.
Fig. 2. The effects of 18 tested phenolic compounds on mycelial growth of Botrytis cinerea. (A) Hierarchical clustering represents effects of the 18 phenolic compounds was performed using mycelial growth measurement at 200 mg L−1, based on the results in Table 2, with the color proportional to the effect of the molecule and the group of compounds belong as illustrated. (B) B. cinerea mycelia TGM grown on PDA medium without (control) or with pterostilbene (200 mg L−1 and 400 mg L−1). Images were taken 72 h after inoculation on plates.
in inhibiting B. cinerea growth in our assessment. In agreement with a previous investigation (Annie and Jean-Jacques, 2003), our study showed that phenolic acids with methoxy (–OCH3) substitution (e.g. sinapic and ferulic acids) were less effective in inhibiting B. cinerea growth than phenolic acids without methoxy substitution (e.g. ρ-coumaric acid). Among the flavonoids, different subgroups compounds including flavones (luteolin and apigenin), flavonols (quercetin and kaempferol), flavanones (naringenin), flavanols (catechin) were tested for their antifungal activities. Naringenin clustered with highly antifungal phenolic (pterostilbene) for its remarkable inhibitory activity: it reduced the growth of GBR, GBW, BGM and TGM by 41.2%, 36.8%, 37.3% and 37.7%, respectively. Except for naringenin, other flavoniod compounds showed either none inhibitory or only strain-specific inhibitory activity,
strains, and seven showed differential effects among B. cinerea strains. In the hierarchical cluster, eight molecules were grouped together based on their significant antifungal activity on all tested strains (Fig. 2A). The compound with highest antifungal activity was pterostilbene, a stilbene of grapevine leaves, which reduced the growth of four target strains to about 4.0–4.8-fold (Fig. 2B). Phenolic acids can be distinguished in two classes depending on their structure: derivatives of benzoic acid (Hydroxybenzoic acids, HBA) and derivatives of cinnamic acid (Hydroxycinnamic acids, HCA) (Lafay and Gil-Izquierdo, 2008). The main HBA are gallic, protocatechuic, ellagic and 4-hydrobenzoic acids (Fig. 1). In our study, gallic acid and protocatechuic acid were tested and none of them showed inhibitory activity (Table 2). HCA are mainly represented by caffeic, ferulic, sinapic and ρ-coumaric acids (Fig. 1), which were more effective 110
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Seven polyphenols (naringenin, coumarin, resveratrol, ferulic acid, catechol, piceatannol and pterostilbene) were chosen to assess the IC50 toward B. cinerea strain TGM because of their highly effective against the four tested strains at 200 mg L−1. Data in Table 3 confirms that pterostilbene with lower IC50 (246 mg L−1) was much more active on B. cinerea than the other phenolic compounds and this may be related to the higher hydrophobicity that increase diffusion through fungal membranes. According to Caruso et al. (2011), there was a positive correlation between antifungal activity of phenolics and their hydrophobicity, which can be evaluated through LogP values. In our study, this correlation was demonstrated, and molecules with a high LogP value showed higher antifungal activities against B. cinerea. For example, stilbenes including pterostilbene, piceatannol and resveratrol, with a LogP value of 4.1, 2.7 and 3.0, respectively, slightly higher than other tested phenolics, showed stronger inhibitory activity on mycelial growth. Catechol displayed negative correlation among antifungal activity in vitro and hydrophobicity, whereas the positive correlation between hydrophobicity and its efficacy on the control of gray mold in vivo proved the importance of hydrophobicity on increasing action activity.
Table 3 IC50 of active phenolic compounds toward tested strains. Phenolic compounds
LogPa
IC50b ± SD (mg L−1)
Naringenin Coumarin Resveratrol Ferulic acid Catechol Piceatannol Pterostilbene
2.6 1.4 3.0 1.0 0.8 2.7 4.1
502 539 438 629 275 486 246
± ± ± ± ± ± ±
7.3 5.9 8.4 9.7 4.3 7.2 7.6
a Estimated octanol/water partition coefficient (LogP), calculated using Advanced Chemistry Development (ACD/Labs) Software V9.04 (1994–2010 ACD/Labs), as stored in SciFinder. b Determination of half maximal inhibitory concentration (IC50), based on colony diameter measurements after 72 h of incubation.
or even growth-promoting activity (Fig. 2A, Table 2). The antimicrobial activity of flavonoids is confirmed to be dependent on their structure and among the flavonoids, flavanones showed the strongest antifungal effects (Mierziak et al., 2014; Seleem et al., 2017). Stilbenes are derived from phenylpropanoid pathway, like flavonoids and phenolic acids, while it exhibited stronger inhibitory activities than the latter two types of compounds. Most stilbenes are derivatives of the basic unit trans-resveratrol, which have two aromatic rings connected by a methylene bridge backbone (Chong et al., 2009). In our study, simple stilbenes including resveratrol (3,4′,5-trihydroxystilbene, a major stilbene enriched in grapevine), pterostilbene (3′,5′dimethoxy-resveratrol) and piceatannol (3,3′,4,5′-tetrahydroxy-stilbene) were tested. Our results confirm previous reports, in which stilbenes showed highest antifungal activity among the tested compounds (Flamini et al., 2013; Chalal et al., 2014). Furthermore, pterostilbene, with two methoxy groups had the highest inhibitory activity on B. cinerea among the three tested simple stilbenes (Fig. 2). However, piceid, a glycosylated derivative of resveratrol (Fig. 1), did not cluster with the other tested stilbenes and displayed a weak inhibitory effect, possibly due to the increasing stability and solubility resulted from glycosylation (Morales et al., 1998; Lambert et al., 2012).
3.2. Impact of phenolic on B. cinerea mycelia Microscopic examination of phenolic-treated B. cinerea mycelia were performed to visualize morphological changes and/or structural alterations induced by the chemicals. The untreated hyphae were tubular, regular and homogeneous morphology as showed in Fig. 3A andB, while the pterostilbene-treated mycelia appeared malformed and swollen (Fig. 3D and E). This result was consistent with the previous observation, which reported that resveratrol resulted in cytological changes in B. cinerea (Adrian et al., 1997). This experiment confirmed that phenolics can exert inhibitory activity on fungal growth by increasing cell permeabilization, as also reported by Luo et al. (2016). Another cellular changes caused by pterostilbene was the increase of vesicles and big vacuoles in the mycelia (Fig. 3C, D and F), which indicated the antifungal mechanism of pterostilbene may related to membrane disruption and/or cell growth hindrance, and of course of
Fig. 3. Effect of pterostilbene on Botrytis cinerea mycelial morphology. Untreated B. cinerea mycelia (A, B) or B. cinerea mycelia treated with 200 mg L−1 pterostilbene for 72 h (C-F) were observed and imaged by optical microscopy equipped with Nikon DS-Ri1 camera.
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Fig. 4. Effects of phenolic compounds on conidial germination of Botrytis cinerea. (A) Mean values( ± SD) presents conidial germination rate calculated from three replicates. Same set of columns followed by different letters indicate significant (P < 0.05) difference between two comparing sets, according to Duncan’s multiple range test. (B) Conidial germination of B. cinerea was examined and imaged under untreated condition (control–0 h and control–7 h, representing pre-cultured for 0 and 7 h respectively) or treated with different concentration of pterostilbene for 7 h.
interests for the future research.
pterostilbene completely inhibited the germination of B. cinerea conidia at 50 mg L−1, and the other compounds at the same concentration inhibited 30%–75% of conidial germination at the same concentration. At a concentration of 10 mg L−1, pterostilbene still showed strong inhibition on B. cinerea conidial germination, of 82%. The second strong tested antimicrobial compound was piceatannol, inhibiting conidial
3.3. Inhibition of conidial germination Seven compounds with significant antifungal activity (Table 3) were evaluated for their effects on conidial germination. As shown in Fig. 4A,
Fig. 5. Gray mold on table grapes control by different phenolic compounds. Disease incidence (A) and lesion diameter (B) was quantified and presented as bar charts. (C) Harvested grape berries were treated with phenolic compounds and inoculated with B. cinerea, then kept at 22 °C. Symptoms were assessed and photographed 9 days post inoculation. Same set of columns followed by different letters indicate significant (P < 0.05) differences according to Duncan’s multiple range test.
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germination of 56%. Some conidia with brown coloration were found following application of phenolic compounds (50 mg L−1), but not in the control (Fig. 4B). Brown pigmentation as described above was reported by Adrian et al. (1998), and it was thought related to the oxidation of phenolic compounds mediated by laccase. Fungal laccase is multicopper phenol oxidase that protect the fungus from the host secondary metabolites, such as phytoalexins, during fungal invasion and plays an important role in the fungal pathogenicity (Guetsky et al., 2005; Sansone et al., 2011). B. cinerea is known to be able to detoxify phytoalexins via laccase-mediated degradation of the secondary metabolite resveratrol (Schouten et al., 2002). Therefore, the laccase of B. cinerea is of potential as a target for postharvest control of gray mold.
C.A., Packer, L. (Eds.), Flavonoids in Health and Disease, 2nd ed. Marcel Dekker Inc., New York, pp. 17–20. Aziz, A., Verhagen, B., Magnin-Robert, M., Couderchet, M., Clément, C., Jeandet, P., Trotel-Aziz, P., 2016. Effectiveness of beneficial bacteria to promote systemic resistance of grapevine to gray mold as related to phytoalexin production in vineyards. Plant Soil 405, 141–153. Caruso, F., Mendoza, L., Castro, P., Cotoras, M., Aguirre, M., Matsuhiro, B., Isaacs, M., Rossi, M., Viglianti, A., Antonioletti, R., 2011. Antifungal activity of resveratrol against Botrytis cinerea is improved using 2-furyl derivatives. PLoS One 6, e25421. Chalal, M., Klinguer, A., Echairi, A., Meunier, P., Vervandier-Fasseur, D., Adrian, M., 2014. Antimicrobial activity of resveratrol analogues. Molecules 19, 7679–7688. Chong, J., Poutaraud, A., Hugueney, P., 2009. Metabolism and roles of stilbenes in plants. Plant Sci. 177, 143–155. De Hoon, M.J.L., Imoto, S., Nolan, J., Miyano, S., 2004. 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Mendoza, L., Yanez, K., Vivanco, M., Melo, R., Cotoras, M., 2013. Characterization of extracts from winery by-products with antifungal activity against Botrytis cinerea. Ind. Crop. Prod. 43, 360–364. Mierziak, J., Kostyn, K., Kulma, A., 2014. Flavonoids as important molecules of plant interactions with the environment. Molecules 19, 16240–16265. Mlikota Gabler, F., Smilanick, J.L., Mansour, M., Ramming, D.W., Mackey, B.E., 2003. Correlations of morphological, anatomical, and chemical features of grape berries with resistance to Botrytis cinerea. Phytopathology 93, 1263–1273. Morales, M., Bru, R., García-Carmona, F., Ros Barceló, A., Pedreño, M.A., 1998. Effect of dimethyl-β-cyclodextrins on resveratrol metabolism in Gamay grapevine cell cultures before and after inoculation with shape Xylophilus ampelinus. Plant Cell. Tiss. Org. Cult. 53, 179–187. Page, R.D.M., 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12, 357–358. Parafati, L., Vitale, A., Restuccia, C., Cirvilleri, G., 2015. Biocontrol ability and action mechanism of food-isolated yeast strains against Botrytis cinerea causing post-harvest bunch rot of table grape. Food Microbiol. 47, 85–92. Piotrowska, H., Kucinska, M., Murias, M., 2012. Biological activity of piceatannol: leaving the shadow of resveratrol. Mutat. Res. 750, 60–82. Pizzolitto, R.P., Barberis, C.L., Dambolena, J.S., Herrera, J.M., Zunino, M.P., Magnoli, C.E., Rubinstein, H.R., Zygadlo, J.A., Dalcero, A.M., 2015. Inhibitory effect of natural phenolic compounds on Aspergillus parasiticus growth. J. Chem. http://dx.doi.org/10. 1155/2015/547925. Romanazzi, G., Gabler, F.M., Smilanick, J.L., 2006. Preharvest chitosan and postharvest UV irradiation treatments suppress gray mold of table grapes. Plant Dis. 90, 445–450. Romanazzi, G., Lichter, A., Gabler, F.M., Smilanick, J.L., 2012. Recent advances on the use of natural and safe alternatives to conventional methods to control postharvest gray mold of table grapes. Postharvest Biol. Technol. 63, 141–147. Romanazzi, G., Smilanick, J.L., Feliziani, E., Droby, S., 2016a. Integrated management of postharvest gray mold on fruit crops. Postharvest Biol. Technol. 113, 69–76. Romanazzi, G., Sanzani, S.M., Bi, Y., Tian, S., Gutiérrez Martínez, P., Alkan, N., 2016b. Induced resistance to control postharvest decay of fruit and vegetables. Postharvest
3.4. In vivo assessment of anti-fungal activity for selected phenolics Seven phenolic compounds showed strong inhibitory effects on B. cinerea growth and conidial germination in vitro, then their effectiveness on gray mold incidence and severity on grape berries was tested. In the seven tested phenolics, pterostilbene and piceatannol treatment significantly prevented the decay of Mare’s milk grapes, while the other phenolics showed little or none effects on disease control (Fig. 5). After 9 days storage at 22 °C, pterostilbene completely inhibited gray mold, and disease incidence in piceatannol-treated grape berries was reduced to 75% compared to the control (Fig. 5A). Lesion diameter of piceatannol or pterostilbene treated grape berries were also significantly reduced compared to the control (Fig. 5B). While the control berries had apparent mycelial biomass beyond the wounded area, grape berries treated with piceatannol or pterostilbene just displayed minimal browning around the inoculation site (wound) and with little mycelia (Fig. 5C). This can be explained by the antifungal activities and antioxidant capacities of pterostilbene and piceatannol (Piotrowska et al., 2012). Overall, our results provided evidence that piceatannol and pterostilbene were effective in the control of postharvest gray mold of table grapes. 4. Conclusions This study is the first systematic investigation on the efficacy of bioactive phenolics on gray mold of table grapes. Our results demonstrated that piceatannol and pterostilbene had strongest activity on B. cinerea, not only greatly inhibited the mycelia growth and conidial germination in vitro, but also exhibit a strong efficacy on preventing or reducing gray mold in “Mare’s milk” table grapes in vivo. Further translational trials are needed to confirm their activity on a larger scale, and their action mechanism also need to be explored. It is of great potential, for natural compounds to be used as alternative strategy to traditional fungicides in the control of postharvest gray mold of table grapes. Acknowledgements This research was supported by the earmarked grants for China agriculture research system (CARS-29-bc) and (CARS-33-11). References Adrian, M., Jeandet, P., 2012. Effects of resveratrol on the ultrastructure of Botrytis cinerea conidia and biological significance in plant/pathogen interactions. Fitoterapia 83, 1345–1350. Adrian, M., Jeandet, P., Veneau, J., Weston, L.A., Bessis, R., 1997. Biological activity of resveratrol, a stilbenic compound from grapevines, against Botrytis cinerea, the causal agent for gray mold. J. Chem. Ecol. 23, 1689–1702. Adrian, M., Rajaei, H., Jeandet, P., Veneau, J., Bessis, R., 1998. Resveratrol oxidation in Botrytis c inerea conidia. Phytopathology 88, 472–476. Ahuja, I., Kissen, R., Bones, A.M., 2012. Phytoalexins in defense against pathogens. Trends Plant Sci. 17, 73–90. Annie, F., Jean-Jacques, M., 2003. Phenolic acids in fruits and vegetables. In: Rice-Evans,
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76–83. Wang, K., Liao, Y., Kan, J., Han, L., Zheng, Y., 2015. Response of direct or priming defense against Botrytis cinerea to methyl jasmonate treatment at different concentrations in grape berries. Int. J. Food Microbiol. 194, 32–39. Wang, M., Jiang, N., Wang, Y., Jiang, D.M., Feng, X.Y., 2017. Characterization of phenolic compounds from early and late ripening sweet cherries and their antioxidant and antifungal activities. J. Agric. Food Chem. 65, 5413–5420. Youssef, K., Roberto, S.R., 2014. Applications of salt solutions before and after harvest affect the quality and incidence of postharvest gray mold of ‘Italia’ table grapes. Postharvest Biol. Technol. 87, 95–102. Zheng, C.L., Choquer, M., Zhang, B., Ge, H., Hu, S.N., Ma, H.Q., Chen, S.W., 2011. LongSAGE gene-expression profiling of Botrytis cinerea germination suppressed by resveratrol, the major grapevine phytoalexin. Fungal Biol. 115, 815–832.
Biol. Technol. 122, 82–94. Sansone, G., Rezza, I., Fernández, G., Calvente, V., Benuzzi, D., Isabel Sanz, M., 2011. Inhibitors of polygalacturonase and laccase of Botrytis cinerea and their application to the control of this fungus. Int. Biodeter. Biodegr. 65, 243–247. Sanzani, S.M., De Girolamo, A., Schena, L., Solfrizzo, M., Ippolito, A., Visconti, A., 2009. Control of Penicillium expansum and patulin accumulation on apples by quercetin and umbelliferone. Eur. Food Res. Technol. 228, 381–389. Sanzani, S.M., Schena, L., Ippolito, A., 2014. Effectiveness of phenolic compounds against citrus green mould. Molecules 19, 12500–12508. Schouten, A., Wagemakers, L., Stefanato, F.L., van der Kaaij, R.M., van Kan, J., 2002. Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase. Mol. Microbiol. 43, 883–894. Seleem, D., Pardi, V., Murata, R.M.A., 2017. Review of flavonoids: a diverse group of natural compounds with anti-Candida albicans activity in vitro. Arch. Oral Biol. 76,
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
In vitro and in vivo effectiveness of phenolic compounds for the control of postharvest gray mold of table grapes
T
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Dandan Xua, Yizhen Dengb, Tingyu Hana, Liqun Jiangb, Pinggen Xib, Qi Wanga, Zide Jiangb, , ⁎ Lingwang Gaoa, a
College of Plant Protection, China Agricultural University, Beijing 100193, China Department of Plant Pathology/Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Antifungal activity Piceatannol Postharvest decay Pterostilbene
As the natural antimicrobial metabolites, phenolic compounds were reported to be effective in the inhibition of phytopathogenic fungi, including the postharvest decay agents. However, comprehensive study on the biological activity of phenolic compounds and their application on controlling postharvest gray mold of table grapes is lacking. In this study, the antifungal effect of 18 natural or synthetic phenolic compounds purchased from commercial suppliers, including simple phenolic, phenolic acids, stilbenes and flavonoids, were determined on four gray mold strains by an in vitro agar dilution assay. Overall, seven phenolic compounds were effective on inhibiting B. cinerea growth and were selected to test their activity on conidial germination as well as in vivo application on grape berries. Pterostilbene showed the highest antifungal activity and greatly reduced the growth of the mycelia, caused hyphae deformation, suppressed conidial germination of B. cinerea, and completely inhibited the germination of conidia at the concentration of 50 mg L−1. Furthermore, treatment of grape berries with pterostilbene and piceatannol significantly reduced the disease incidence and severity. Our results demonstrate the antifungal activity of phenolic compounds and highlight their potentials as an alternative strategy in the control of postharvest gray mold of table grapes.
1. Introduction The fungus Botrytis cinerea causes gray mold, leads to huge postharvest losses and is considered as the main postharvest decay of table grapes (Vitis vinifera) (Romanazzi et al., 2012; Youssef and Roberto, 2014). In many countries, synthetic fungicides are not allowed to use for the control of postharvest decay of table grapes, and only sulfur dioxide (SO2) is permitted to use as an adjuvant. Alternatives to SO2 are urgently required in view of the hazardous effect to human health caused by SO2 residues and the bleaching injuries in berries caused by the application of SO2 (Gándara-Ledezma et al., 2015; Parafati et al., 2015; Romanazzi et al., 2016a). Among numerous unconventional control strategies, the induction of fruit resistance via application of plant or microorganism products with antimicrobial activity and the physical means can be considered, either alone or as a part of an integrated pest management policy (Romanazzi et al., 2016b). Within plant natural products, phenolic compounds, which widely distribute in the tissues of resistant
grapevine cultivars as important secondary metabolites, are thought to be involved in defence response against fungi by forming a chemical barrier that limits pathogen growth and increase plant resistance (Mlikota Gabler et al., 2003; Del Rio et al., 2004; Pizzolitto et al., 2015). The fungus B. cinerea is present in vineyards as part of the environmental microflora, infection of grape often occurs at bloom time, followed by a period of latency, and generally causes disease symptoms when berries begin to ripen and meanwhile phenolic compounds contents changes (Holz et al., 2003; Keller et al., 2003; Liang et al., 2011). Therefore, the phenolic compounds (phytoanticipins) that are constitutively present in grape berries and with potential in inhibiting B. cinerea growth are of great interest. Accumulation of certain phenolics are induced as a response to biotic and abiotic stress, and these metabolites are referred to as phytoalexins (Ahuja et al., 2012). Some phytoalexins have broad spectrum of anti-pathogen activities and their accumulation in plants can promote host defense response, e.g. the accumulation of trans-resveratrol and catechin in grapes exposed to chitosan and UV-C (Romanazzi et al., 2006), and the accumulation of
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Corresponding authors at: College of Plant Protection, China Agricultural University, Beijing 100193, China E-mail addresses: [email protected] (D. Xu), [email protected] (Y. Deng), [email protected] (T. Han), [email protected] (L. Jiang), [email protected] (P. Xi), [email protected] (Q. Wang), [email protected] (Z. Jiang), [email protected] (L. Gao). https://doi.org/10.1016/j.postharvbio.2017.08.019 Received 10 February 2017; Received in revised form 2 August 2017; Accepted 27 August 2017 0925-5214/ © 2017 Published by Elsevier B.V.
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Fig. 1. Structures of the 18 tested phenolic compounds.
biological activity of phenolic compounds against B. cinerea, as well as practical feasibility of their application for protection of table grapes form postharvest gray mold. In this study, 18 phenolic compounds including simple phenolic, phenolic acids, stilbenes, flavonoids that were previously reported to have antimicrobial activity, were selected and evaluated in the aspect of their antifungal activity against B. cinerea, both in vitro and in vivo.
stilbenic phytoalexins (resveratrol and viniferin) in grapevine treated with rhizospheric bacteria (Aziz et al., 2016), associated with the development of resistance against B.cinerea. At present, several phenolic compounds were proved to have antifungal properties against Fusarium graminearum (Gauthier et al., 2016), Alternaria alternata (Wang et al., 2017), Penicillium expansum (Sanzani et al., 2014), Plasmopara viticola (Gabaston et al., 2017) and B. cinerea (Mendoza et al., 2013), making them as good “natural pesticide” candidates for improving plant resistance to phytopathogen. However, little is known about the 107
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compound was determined at 72 h post B. cinerea inoculation. The data were presented with a hierarchical clustering of mycelial growth for each fungus in the presence of 200 mg L−1 phenolic compounds, with the control condition set as 100%, which were organized and visualized using the hierarchical clustering software Gene Cluster 3.0 (De Hoon et al., 2004) and Tree view software (Page, 1996). To assess the absolute effectiveness of some active phenolics on the B. cinerea, the concentration inhibiting 50% of fungal growth (IC50) was measured under various concentrations. Mycelium morphology of tested fungal samples was examined and imaged by optical microscopy (Eclipse Ni, Nikon), equipped with Nikon DS-Ri1 camera. For conidial germination assay, B. cinerea spore suspension was prepared by washing the conidia with PDB (potato dextrose broth medium, 37.5 g potato, 20 g dextrose per liter) and the final concentration was adjusted as 105 conidia per milliliter. Then, equal volume of phenolic compounds or ethanol, as solvent control, was added into the spore suspension (Zheng et al., 2011). All tested samples were kept in dark to protect the phenolics (Adrian and Jeandet, 2012), at 22 °C. Conidial germination was determined by calculating the percentage of germinated spores. Each treatment was repeated three times.
Table 1 Technical facts of compounds used in this study. Phenolic compound
CAS
Catalog number
Source
Resveratrol Luteolin Apigenin Catechol Ferulic acid Catechin Coumarin Caffeic acid Quercetin ρ-coumaric acid Sinapic acid Naringenin Gallic acid Piceid Pterostilbene Kaempferol Protocatechuic acid Piceatannol
501−36-0 491−70-3 520−36-5 120−80-9 537−98-4 154−23-4 91−64-5 331−39-5 117−39-5 501−98-4 530−59-6 480−41-1 149−91-7 65914−17-2 537−42-8 520−18-3 99−50-3 10083−24-6
V900386 79506 M01289 359694 408509 C217500 107521 247526 268623 C9008 397578 393137 210917 296286 455753 258643 223812 P1928
Sigma-Aldrich Fluorochem Ltd Fluorochem Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd J&K Scientific Ltd
2. Materials and methods
2.5. In vivo bioassays
2.1. Fruit material
The antifungal activity of phenolic compounds against B. cinerea was assessed on grape berries by wounded assay (Sanzani et al., 2009). Two mm deep wounds in the equatorial zone were made by dissected needles and inoculated with 10 μL of phenolic solution (104 mg L−1, 100 μg per wound). After drying at room temperature for approximately 30 min, each wound was inoculated with 10 μL of a 5 × 104 conidia per milliliter suspension of B. cinerea and allowed to air-dry for 1 h. Wounds with equal volume of solvent and inoculated with the B. cinerea severed as a control. The experiment repeated twice with three replicates, each consisting of 45 berries per treatment. All treated berries were sealed in plastic containers and kept at 22 °C in dark and approximately 80% relative humidity for 9 days. Disease symptom was examined by visual evaluation, in which grape berries showing apparent water-soaked lesions with mycelia and sporulation beyond the wounded area were considered as infected (Wang et al., 2015). Disease incidence was calculated as percentage of infected berries and the rot lesion diameters were recorded.
Fresh table grape berries (cv Mare’s milk) were carefully removed from the rachis, keeping pedicels intact. The berries without visible defects or injuries, and with uniform size and ripening stage were chosen. Before treatments, the selected berries were randomized, surface sterilized in a 2% sodium hypochlorite solution for 2 min, rinsed under running tap water for 1 min, and air-dried at room temperature. 2.2. Plant pathogen B. cinerea isolates TGM and BGM used in this study were kindly provided by the MOA Key Laboratory of Plant Pathology (China Agricultural University, Beijing), and B. cinerea isolates GBR and GBW were obtained from table grape berries with typical gray mold symptoms. All fungal strains were maintained on PDA medium (potato dextrose agar medium, 200 g potato, 20 g dextrose and 15 g agar per liter) at 4 °C until use.
2.6. Statistical analysis 2.3. Chemical reagents Data were statistically analyzed using analysis of variance (ANOVA) and expressed as mean ± standard deviation (SD). The mean separations were analyzed using Duncan’s multiple range tests and differences among different treatments was determined at 5% level (IBM SPSS Statistics 22, USA).
All phenolic compounds (Fig. 1) used in the study had a purity grade higher than 98%. Resveratrol was purchased from Sigma-Aldrich (St. Louis, MO, USA), and apigenin and luteolin were purchased from Fluorochem Ltd (Old Glossop, Derbyshire, UK), the other tested phenolic compounds were purchased from J&K Scientific Ltd (Beijing, China). Detailed information of tested compounds listed in Table 1. All compounds were dissolved in ethanol to prepare stock solutions.
3. Results and discussion 3.1. In vitro assessment of anti-fungal activity for 18 phenolics
2.4. In vitro bioassays The 18 tested phenolic compounds differentially affected fungal growth and sensitivity toward phenolics differed among the four tested strains (Table 2). Six compounds (catechol, resveratrol, coumarin, naringenin, pterostilbene and piceatannol) displayed antifungal activity (growth inhibition more than 20%) and protocatechuic acid enhanced the growth for all tested strains. Kaempferol exhibited no activity (growth affected less than 6%) in all tested strains. However, some phenolic compounds showed differential effects on mycelial growth of the four tested fungal strains, i.e. ferulic acid suppressed B. cinerea isolates GBR, GBW and BGM growth, while enhanced the growth of TGM. Ten out of 18 tested molecules had an inhibitory activity on fungal growth for all the four strains tested, one stimulated growth for the four
The antifungal activity of phenolic compounds was assessed according to Lambert et al. (2012). Phenolic compounds were added to the cooled PDA medium respectively, with an equal volume of ethanol added to the PDA medium as solvent control. Plug inocula of 6 mm diameter were cut from the actively growing fungal cultures and then placed at the center of PDA plate, with or without phenolic supplements. The cultures were incubated at 22 °C in the dark. Colony diameter (in mm) was measured at 24-h intervals for five consecutive days. Each phenolic compound was assessed on the four isolates. Four independent replicates were performed for each instance and experiment was carried out twice. The percentage of growth inhibition or induction for each phenolic 108
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Table 2 Effect of phenolic compounds on the colony diameter of B.cinereaTable 2 (Continued). Phenolic compounds
Concentration (mg L−1)
Mycelial growth of different tested strains (mm) GBR
GBW
BGM
TGM
ρ-coumaric acid
0 50 100 200
65.4 44.3 38.8 37.0
± ± ± ±
3.4 1.5 0.8 1.0
64.9 56.5 55.5 53.0
± ± ± ±
2.5 1.3 0.5 1.0
63.6 56.5 54.7 46.1
± ± ± ±
2.4 1.3 1.2 0.1
69.6 54.8 50.9 49.9
± ± ± ±
0.7 0.8 0.8 1.5
Resveratrol
0 50 100 200
63.5 62.5 57.7 51.8
± ± ± ±
1.3 1.5 1.1 0.7
65.3 59.2 58.5 46.2
± ± ± ±
1.1 1.0 2.5 0.4
72.4 67.5 63.3 54.7
± ± ± ±
0.8 1.3 1.5 1.6
71.3 64.3 55.2 44.1
± ± ± ±
0.9 2.1 2.7 1.1
Sinapic acid
0 50 100 200
69.0 63.3 61.8 60.9
± ± ± ±
1.4 1.3 2.0 1.2
64.9 61.8 60.3 58.8
± ± ± ±
2.5 0.4 1.2 0.9
66.3 60.0 59.8 57.3
± ± ± ±
0.4 1.0 1.0 0.4
69.6 60.4 59.3 56.8
± ± ± ±
0.7 0.6 0.8 0.9
Coumarin
0 50 100 200
67.2 66.1 62.8 55.1
± ± ± ±
1.2 1.7 3.9 0.9
65.8 62.8 58.8 53.1
± ± ± ±
1.1 0.6 0.6 0.8
60.8 56.8 54.5 53.6
± ± ± ±
1.1 2.4 3.0 0.4
61.8 57.6 55.8 49.1
± ± ± ±
0.8 0.5 1.8 1.7
Catechol
0 50 100 200
59.4 55.3 49.6 38.2
± ± ± ±
0.8 0.3 1.2 0.7
48.9 45.7 38.3 26.8
± ± ± ±
1.9 3.4 2.3 3.5
57.2 56.0 52.5 40.0
± ± ± ±
1.5 2.2 0.9 0.4
57.9 57.0 44.3 34.9
± ± ± ±
1.7 3.0 3.6 3.3
Piceatannol
0 50 100 200
52.3 46.0 39.5 39.1
± ± ± ±
3.2 0.9 1.3 0.7
64.5 54.5 48.3 38.7
± ± ± ±
0.7 2.6 0.8 2.3
65.7 63.8 54.5 52.3
± ± ± ±
4.2 2.0 2.2 1.8
57.0 52.3 49.8 40.5
± ± ± ±
0.7 1.9 2.9 4.6
Naringenin
0 50 100 200
59.4 57.7 46.8 34.9
± ± ± ±
0.8 2.8 2.3 1.5
48.9 46.9 40.4 30.9
± ± ± ±
1.9 1.2 0.9 0.5
57.2 51.8 43.1 35.8
± ± ± ±
1.5 0.5 2.2 1.2
57.9 42.3 41.4 36.1
± ± ± ±
1.7 2.6 4.1 2.1
Pterostilbene
0 50 100 200
58.5 33.8 31.3 30.3
± ± ± ±
0.9 0.5 0.4 0.3
56.4 35.7 33.8 32.6
± ± ± ±
2.0 0.5 1.3 1.0
66.8 42.3 37.9 35.1
± ± ± ±
1.8 2.4 2.0 2.7
53.2 33.9 31.9 31.8
± ± ± ±
1.3 2.8 1.4 1.8
Kaempferol
0 50 100 200
66.3 63.0 63.3 62.8
± ± ± ±
1.4 1.5 0.7 0.8
61.9 62.3 60.2 57.9
± ± ± ±
1.4 1.0 5.8 4.4
56.7 57.0 54.6 56.4
± ± ± ±
0.6 3.5 1.4 1.1
50.3 48.3 47.0 45.3
± ± ± ±
2.1 2.5 1.7 1.3
Ferulic acid
0 50 100 200
63.5 60.5 55.7 50.0
± ± ± ±
1.3 2.3 1.5 0.9
63.2 59.8 56.9 50.3
± ± ± ±
3.7 1.8 0.8 0.8
82.7 76.0 66.0 58.8
± ± ± ±
2.1 3.5 2.6 0.9
47.0 65.7 58.9 52.7
± ± ± ±
2.8 1.5 1.0 1.5
Caffeic acid
0 50 100 200
67.2 65.2 61.5 58.7
± ± ± ±
1.2 2.5 1.0 2.4
65.8 61.5 60.3 59.1
± ± ± ±
1.1 1.0 2.1 1.0
60.8 59.1 57.5 59.3
± ± ± ±
1.1 1.0 1.5 1.2
61.8 57.7 59.0 60.2
± ± ± ±
0.8 0.8 1.3 2.3
Luteolin
0 50 100 200
66.3 57.8 57.5 57.3
± ± ± ±
1.4 2.2 1.3 0.8
61.9 59.1 58.9 58.8
± ± ± ±
1.4 6.6 2.6 1.4
56.7 57.2 56.5 54.5
± ± ± ±
0.6 1.1 4.8 1.3
50.3 53.3 53.8 50.3
± ± ± ±
2.1 1.9 2.1 1.4
Apigenin
0 50 100 200
66.3 59.6 57.4 56.8
± ± ± ±
1.4 2.3 0.8 4.9
62.3 61.9 59.3 57.7
± ± ± ±
1.4 1.4 1.9 2.5
56.7 61.8 59.4 52.5
± ± ± ±
0.6 2.0 5.6 0.9
53.3 49.0 50.3 50.0
± ± ± ±
1.5 2.6 2.1 0.9
Catechin
0 50 100 200
67.3 63.5 67.7 72.0
± ± ± ±
1.6 1.5 2.4 1.0
72.6 76.9 78.4 76.5
± ± ± ±
0.5 0.5 1.5 1.3
68.7 68.0 71.2 76.8
± ± ± ±
1.2 2.9 0.8 0.7
61.2 54.1 54.4 57.3
± ± ± ±
1.3 1.2 0.8 2.2
Quercetin
0 50 100 200
61.8 59.8 58.2 57.0
± ± ± ±
1.8 0.6 0.7 0.5
59.8 60.2 60.8 61.9
± ± ± ±
2.1 1.3 1.2 0.5
60.7 65.4 67.3 69.4
± ± ± ±
2.3 1.7 2.1 1.2
59.3 60.3 60.9 61.6
± ± ± ±
1.3 1.5 2.3 1.6
Gallic acid
0 50 100 200
59.4 60.7 57.6 56.3
± ± ± ±
0.8 0.6 4.1 4.3
48.9 53.8 55.4 53.7
± ± ± ±
1.9 0.8 1.2 0.3
57.2 61.7 63.7 62.8
± ± ± ±
1.5 5.1 3.3 1.4
57.9 61.7 60.2 61.7
± ± ± ±
1.7 3.3 0.6 3.4
(continued on next page)
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Table 2 (continued) Phenolic compounds
Concentration (mg L−1)
Mycelial growth of different tested strains (mm) GBR
GBW
BGM
TGM
Piceid
0 50 100 200
58.5 60.6 61.3 57.9
± ± ± ±
0.9 1.0 1.0 3.8
56.4 56.3 59.8 60.1
± ± ± ±
2.0 0.8 2.0 1.3
66.8 64.7 68.3 71.1
± ± ± ±
1.8 4.5 1.9 2.0
53.2 56.4 57.1 57.3
± ± ± ±
1.3 4.3 2.1 0.9
Protocatechuic acid
0 50 100 200
50.8 55.6 58.0 58.5
± ± ± ±
2.0 1.0 0.9 2.0
26.2 29.3 32.0 32.8
± ± ± ±
0.3 1.6 0.5 2.1
63.8 62.0 64.3 65.8
± ± ± ±
1.4 0.5 0.7 0.3
51.7 58.0 59.3 61.3
± ± ± ±
1.2 0.9 3.2 0.9
Each value is the mean colony diameter ( ± SD) after 72 h of incubation.
Fig. 2. The effects of 18 tested phenolic compounds on mycelial growth of Botrytis cinerea. (A) Hierarchical clustering represents effects of the 18 phenolic compounds was performed using mycelial growth measurement at 200 mg L−1, based on the results in Table 2, with the color proportional to the effect of the molecule and the group of compounds belong as illustrated. (B) B. cinerea mycelia TGM grown on PDA medium without (control) or with pterostilbene (200 mg L−1 and 400 mg L−1). Images were taken 72 h after inoculation on plates.
in inhibiting B. cinerea growth in our assessment. In agreement with a previous investigation (Annie and Jean-Jacques, 2003), our study showed that phenolic acids with methoxy (–OCH3) substitution (e.g. sinapic and ferulic acids) were less effective in inhibiting B. cinerea growth than phenolic acids without methoxy substitution (e.g. ρ-coumaric acid). Among the flavonoids, different subgroups compounds including flavones (luteolin and apigenin), flavonols (quercetin and kaempferol), flavanones (naringenin), flavanols (catechin) were tested for their antifungal activities. Naringenin clustered with highly antifungal phenolic (pterostilbene) for its remarkable inhibitory activity: it reduced the growth of GBR, GBW, BGM and TGM by 41.2%, 36.8%, 37.3% and 37.7%, respectively. Except for naringenin, other flavoniod compounds showed either none inhibitory or only strain-specific inhibitory activity,
strains, and seven showed differential effects among B. cinerea strains. In the hierarchical cluster, eight molecules were grouped together based on their significant antifungal activity on all tested strains (Fig. 2A). The compound with highest antifungal activity was pterostilbene, a stilbene of grapevine leaves, which reduced the growth of four target strains to about 4.0–4.8-fold (Fig. 2B). Phenolic acids can be distinguished in two classes depending on their structure: derivatives of benzoic acid (Hydroxybenzoic acids, HBA) and derivatives of cinnamic acid (Hydroxycinnamic acids, HCA) (Lafay and Gil-Izquierdo, 2008). The main HBA are gallic, protocatechuic, ellagic and 4-hydrobenzoic acids (Fig. 1). In our study, gallic acid and protocatechuic acid were tested and none of them showed inhibitory activity (Table 2). HCA are mainly represented by caffeic, ferulic, sinapic and ρ-coumaric acids (Fig. 1), which were more effective 110
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Seven polyphenols (naringenin, coumarin, resveratrol, ferulic acid, catechol, piceatannol and pterostilbene) were chosen to assess the IC50 toward B. cinerea strain TGM because of their highly effective against the four tested strains at 200 mg L−1. Data in Table 3 confirms that pterostilbene with lower IC50 (246 mg L−1) was much more active on B. cinerea than the other phenolic compounds and this may be related to the higher hydrophobicity that increase diffusion through fungal membranes. According to Caruso et al. (2011), there was a positive correlation between antifungal activity of phenolics and their hydrophobicity, which can be evaluated through LogP values. In our study, this correlation was demonstrated, and molecules with a high LogP value showed higher antifungal activities against B. cinerea. For example, stilbenes including pterostilbene, piceatannol and resveratrol, with a LogP value of 4.1, 2.7 and 3.0, respectively, slightly higher than other tested phenolics, showed stronger inhibitory activity on mycelial growth. Catechol displayed negative correlation among antifungal activity in vitro and hydrophobicity, whereas the positive correlation between hydrophobicity and its efficacy on the control of gray mold in vivo proved the importance of hydrophobicity on increasing action activity.
Table 3 IC50 of active phenolic compounds toward tested strains. Phenolic compounds
LogPa
IC50b ± SD (mg L−1)
Naringenin Coumarin Resveratrol Ferulic acid Catechol Piceatannol Pterostilbene
2.6 1.4 3.0 1.0 0.8 2.7 4.1
502 539 438 629 275 486 246
± ± ± ± ± ± ±
7.3 5.9 8.4 9.7 4.3 7.2 7.6
a Estimated octanol/water partition coefficient (LogP), calculated using Advanced Chemistry Development (ACD/Labs) Software V9.04 (1994–2010 ACD/Labs), as stored in SciFinder. b Determination of half maximal inhibitory concentration (IC50), based on colony diameter measurements after 72 h of incubation.
or even growth-promoting activity (Fig. 2A, Table 2). The antimicrobial activity of flavonoids is confirmed to be dependent on their structure and among the flavonoids, flavanones showed the strongest antifungal effects (Mierziak et al., 2014; Seleem et al., 2017). Stilbenes are derived from phenylpropanoid pathway, like flavonoids and phenolic acids, while it exhibited stronger inhibitory activities than the latter two types of compounds. Most stilbenes are derivatives of the basic unit trans-resveratrol, which have two aromatic rings connected by a methylene bridge backbone (Chong et al., 2009). In our study, simple stilbenes including resveratrol (3,4′,5-trihydroxystilbene, a major stilbene enriched in grapevine), pterostilbene (3′,5′dimethoxy-resveratrol) and piceatannol (3,3′,4,5′-tetrahydroxy-stilbene) were tested. Our results confirm previous reports, in which stilbenes showed highest antifungal activity among the tested compounds (Flamini et al., 2013; Chalal et al., 2014). Furthermore, pterostilbene, with two methoxy groups had the highest inhibitory activity on B. cinerea among the three tested simple stilbenes (Fig. 2). However, piceid, a glycosylated derivative of resveratrol (Fig. 1), did not cluster with the other tested stilbenes and displayed a weak inhibitory effect, possibly due to the increasing stability and solubility resulted from glycosylation (Morales et al., 1998; Lambert et al., 2012).
3.2. Impact of phenolic on B. cinerea mycelia Microscopic examination of phenolic-treated B. cinerea mycelia were performed to visualize morphological changes and/or structural alterations induced by the chemicals. The untreated hyphae were tubular, regular and homogeneous morphology as showed in Fig. 3A andB, while the pterostilbene-treated mycelia appeared malformed and swollen (Fig. 3D and E). This result was consistent with the previous observation, which reported that resveratrol resulted in cytological changes in B. cinerea (Adrian et al., 1997). This experiment confirmed that phenolics can exert inhibitory activity on fungal growth by increasing cell permeabilization, as also reported by Luo et al. (2016). Another cellular changes caused by pterostilbene was the increase of vesicles and big vacuoles in the mycelia (Fig. 3C, D and F), which indicated the antifungal mechanism of pterostilbene may related to membrane disruption and/or cell growth hindrance, and of course of
Fig. 3. Effect of pterostilbene on Botrytis cinerea mycelial morphology. Untreated B. cinerea mycelia (A, B) or B. cinerea mycelia treated with 200 mg L−1 pterostilbene for 72 h (C-F) were observed and imaged by optical microscopy equipped with Nikon DS-Ri1 camera.
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Fig. 4. Effects of phenolic compounds on conidial germination of Botrytis cinerea. (A) Mean values( ± SD) presents conidial germination rate calculated from three replicates. Same set of columns followed by different letters indicate significant (P < 0.05) difference between two comparing sets, according to Duncan’s multiple range test. (B) Conidial germination of B. cinerea was examined and imaged under untreated condition (control–0 h and control–7 h, representing pre-cultured for 0 and 7 h respectively) or treated with different concentration of pterostilbene for 7 h.
interests for the future research.
pterostilbene completely inhibited the germination of B. cinerea conidia at 50 mg L−1, and the other compounds at the same concentration inhibited 30%–75% of conidial germination at the same concentration. At a concentration of 10 mg L−1, pterostilbene still showed strong inhibition on B. cinerea conidial germination, of 82%. The second strong tested antimicrobial compound was piceatannol, inhibiting conidial
3.3. Inhibition of conidial germination Seven compounds with significant antifungal activity (Table 3) were evaluated for their effects on conidial germination. As shown in Fig. 4A,
Fig. 5. Gray mold on table grapes control by different phenolic compounds. Disease incidence (A) and lesion diameter (B) was quantified and presented as bar charts. (C) Harvested grape berries were treated with phenolic compounds and inoculated with B. cinerea, then kept at 22 °C. Symptoms were assessed and photographed 9 days post inoculation. Same set of columns followed by different letters indicate significant (P < 0.05) differences according to Duncan’s multiple range test.
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germination of 56%. Some conidia with brown coloration were found following application of phenolic compounds (50 mg L−1), but not in the control (Fig. 4B). Brown pigmentation as described above was reported by Adrian et al. (1998), and it was thought related to the oxidation of phenolic compounds mediated by laccase. Fungal laccase is multicopper phenol oxidase that protect the fungus from the host secondary metabolites, such as phytoalexins, during fungal invasion and plays an important role in the fungal pathogenicity (Guetsky et al., 2005; Sansone et al., 2011). B. cinerea is known to be able to detoxify phytoalexins via laccase-mediated degradation of the secondary metabolite resveratrol (Schouten et al., 2002). Therefore, the laccase of B. cinerea is of potential as a target for postharvest control of gray mold.
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3.4. In vivo assessment of anti-fungal activity for selected phenolics Seven phenolic compounds showed strong inhibitory effects on B. cinerea growth and conidial germination in vitro, then their effectiveness on gray mold incidence and severity on grape berries was tested. In the seven tested phenolics, pterostilbene and piceatannol treatment significantly prevented the decay of Mare’s milk grapes, while the other phenolics showed little or none effects on disease control (Fig. 5). After 9 days storage at 22 °C, pterostilbene completely inhibited gray mold, and disease incidence in piceatannol-treated grape berries was reduced to 75% compared to the control (Fig. 5A). Lesion diameter of piceatannol or pterostilbene treated grape berries were also significantly reduced compared to the control (Fig. 5B). While the control berries had apparent mycelial biomass beyond the wounded area, grape berries treated with piceatannol or pterostilbene just displayed minimal browning around the inoculation site (wound) and with little mycelia (Fig. 5C). This can be explained by the antifungal activities and antioxidant capacities of pterostilbene and piceatannol (Piotrowska et al., 2012). Overall, our results provided evidence that piceatannol and pterostilbene were effective in the control of postharvest gray mold of table grapes. 4. Conclusions This study is the first systematic investigation on the efficacy of bioactive phenolics on gray mold of table grapes. Our results demonstrated that piceatannol and pterostilbene had strongest activity on B. cinerea, not only greatly inhibited the mycelia growth and conidial germination in vitro, but also exhibit a strong efficacy on preventing or reducing gray mold in “Mare’s milk” table grapes in vivo. Further translational trials are needed to confirm their activity on a larger scale, and their action mechanism also need to be explored. It is of great potential, for natural compounds to be used as alternative strategy to traditional fungicides in the control of postharvest gray mold of table grapes. Acknowledgements This research was supported by the earmarked grants for China agriculture research system (CARS-29-bc) and (CARS-33-11). References Adrian, M., Jeandet, P., 2012. Effects of resveratrol on the ultrastructure of Botrytis cinerea conidia and biological significance in plant/pathogen interactions. Fitoterapia 83, 1345–1350. Adrian, M., Jeandet, P., Veneau, J., Weston, L.A., Bessis, R., 1997. Biological activity of resveratrol, a stilbenic compound from grapevines, against Botrytis cinerea, the causal agent for gray mold. J. Chem. Ecol. 23, 1689–1702. Adrian, M., Rajaei, H., Jeandet, P., Veneau, J., Bessis, R., 1998. Resveratrol oxidation in Botrytis c inerea conidia. Phytopathology 88, 472–476. Ahuja, I., Kissen, R., Bones, A.M., 2012. Phytoalexins in defense against pathogens. Trends Plant Sci. 17, 73–90. Annie, F., Jean-Jacques, M., 2003. Phenolic acids in fruits and vegetables. In: Rice-Evans,
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