Exogenous caffeic acid and epicatechin enhance resistance against Botrytis cinerea through activation of the phenylpropanoid pathway in apples

doi:10.1016/j.scienta.2020.109348

Scientia Horticulturae

Volume 268, 27 June 2020, 109348

https://doi.org/10.1016/j.scienta.2020.109348 Get rights and content

Highlights

•
Caffeic acid and Epicatechin treatments were found to inhibit the gray mold of apple by regulating phenylpropane metabolism pathway.
•
Caffeic acid treatment effectively promoted lignin accumulation.
•
Epicatechin significantly increased the accumulation of flavonoids.

Abstract

The gray mold caused by Botrytis cinerea is a major postharvest disease of apple which seriously reduce the commercial value. This study was to evaluate the effectiveness of caffeic acid (CA) and epicatechin (E) on controlling gray mold of ‘Fuji’ apples (Malus domestica Borkh.) and explore its mechanism of action. Two treatment groups were injected with 10 μL CA (300 mg/L) and E (300 mg/L) respectively and distilled water was used as control, after half an hour, inoculated with B. cinerea conidial suspension. The decrease in the incidence and lesion diameters of apples by these two free phenols are shown. CA and E activated peroxidase, polyphenoloxidase, phenylalanine ammonia-lyase, cinnamic acid-4-hydroxylase and 4-coumaric acid-CoA ligase, and also increased the content of total phenols, flavonoids and lignin. CA-treated effectively promoted lignin accumulation, whereas E-treated significantly increased the accumulation of flavonoids. Therefore, these two free phenol treatments effectively inhibit gray mold infection of ‘Fuji’ apples by related effective activation of different branches of the phenylpropanoid metabolism pathway.

Introduction

Early and rapid accumulation of phenolic compounds at infection sites is involved with disease resistance in plant-pathogen interactions (Matern and Kneusel, 1988). The phenylpropanoid pathway plays an important role in plant and fruit disease resistance (Yong-Hong et al., 2012). Phenolics are important secondary metabolites of the phenylpropanoid pathway. Phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydro-xylase (C4H) and 4-coumarate-CoA ligase (4CL) are the first three key enzymes in the phenylpropanoid metabolic pathway and convert phenylalanine to 4-coumaroyl-CoA, which can be used to synthesize phenolic acids, flavonoids, lignins and other phenolics (Bevilaqua et al., 2019; Ferrer et al., 2008). The regulation of phenylpropanoid metabolism promotes the accumulation of phenolic substances and affects the resistance of fruits against pathogens (Deng et al., 2015; Li et al., 2017; Liu et al., 2014). Although studies have shown that after infection with pathogen the increment of total phenol content in resistance plants is higher than that of susceptible varieties, the original content before infection is not necessarily proportional to the disease resistance (Dicko et al., 2005; Slatnar et al., 2010). In addition, Ma et al. (2018) reported that the original content and changing content of the free phenol differs significantly between gray mold-resistant and susceptible cultivars of apple (Ma et al., 2018). The synthesis and accumulation of phenols is considered to be a vital mechanism of the plant stress response, with free phenols the probable foundation of stress response and resistance.

In the preliminary test, several free phenols with antifungal activity against Botrytis cinerea (B. cinerea) were screened out from the main phenols in apple, including caffeic acid and epicatechin. Caffeic acid and epicatechin are found in many species that have known antimicrobial activity (Lima et al., 2016; Prakash et al., 2019). Caffeic acid is one of the common phenolic acid in apples (Ma et al., 2018). As an important synthetic precursor of lignin, exogenous caffeic acid can be channeled into the phenylpropanoid pathway, resulting in an increase of the lignin monomer (Bubna et al., 2011). Flavanols (catechins, epicatechins, proanthocyanidins) in apples are a class of flavonoids. Flavanols chelate metals, which reportedly limit growth of invasive microorganisms by causing severe essential mineral depletion (Aron and Kennedy, 2008). Epicatechin is a synthetic precursor of proanthocyanidins and a decrease in proanthocyanidins levels weakens fruit resistance (Terry et al., 2004). In addition to directly inhibiting the growth of B. cinerea, we hypothesize that these two phenols also play an important role in the disease resistance of apples by affecting phenylpropanoid metabolism.

In this study, exogenous phenol treatment was used to explore the differences in resistance to gray mold, and the differences in key enzymes and metabolites of phenylpropanoid metabolism were also analyzed to reveal the role of caffeic acid and epicatechin in the resistance to gray mold, thereby affording a theoretical basis for further studies on the resistance mechanism of phenolic substances to disease.

Section snippets

Fruit material and fungal pathogen

Fruits of ‘Fuji’ apple (Malus domestica Borkh.) were harvested at commercial orchard from Shannxi Province, China (35.18 °N; 109.58 °E). The fruits were harvested at total soluble solids between 15 ∼ 18 %, packed individually in net bags of foam plastic, and put in the corrugated case. Field heat was removed for 12 h at room temperature and fruit were selected with uniform size, color and ripeness and lacked any physical injuries or apparent infections.

Botrytis cinerea Pers. was provided by the

Effect of two free phenols on incidence and lesion diameter

The incidence and lesion diameters are important indexes to evaluate the effect of fruit resistance. As shown in Fig. 1, the two free phenols had significant inhibitory effects on the incidence and the diameter expansion of lesions in apples inoculated with B. cinerea. 1 d after inoculation, the infection rate of CA and E treatments was 6.7 % and 10 %, respectively, while that of CK was close to 20 %. The lesion diameter of treated groups was significantly smaller than that in CK (p < 0.05)

Discussion

Phenolic compounds are a class of secondary metabolites formed by the Phenylpropanoid metabolism pathway in plants, which have antimicrobial, anti-oxidative and free radical elimination activity, and play an important role in plant disease resistance (Kalinowska et al., 2014; Suárez et al., 2010). Additionally, PPO is involved in the oxidation of polyphenols into quinones, which are compounds that enhance plant disease resistance (Gorny et al., 2002). In recent years, research on the use of

Conclusion

Caffeic acid treatment and epicatechin treatment were found to inhibit effectively the gray mold of ‘Fuji’ apple, and the phenols function by effective activation of the phenylpropanoid metabolism pathway. Caffeic acid treatment effectively promoted lignin accumulation, whereas epicatechin significantly increased the accumulation of flavonoids. Therefore, these two free phenol inhibit gray mold infection of apples by effective activation of different branches of the phenylpropanoid metabolism

CRediT authorship contribution statement

Mengyu Zhang: Conceptualization, Methodology, Software. Dajiang Wang: Data curation, Writing - original draft. Xixi Gao: Visualization, Investigation. Zhengyang Yue: Supervision, Software, Validation. Huiling Zhou: Writing - review & editing.

Declaration of Competing Interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.

Acknowledgment

This study was supported by the Modern Agricultural Industry Technology System of Apple (CHINA), Grant/Award Number: nycylx-08-05-02. The authors thank Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

References (38)

T.H. Attridge et al.
Evidence for a pool of inactive phenylalanine ammonia-lyase in Cucumis sativus seedlings
Phytochemistry
(1973)
J.M. Bevilaqua et al.
Exogenous application of rosmarinic acid improves saccharification without affecting growth and lignification of maize
Plant Physiol. Biochem.
(2019)
G.A. Bubna et al.
Exogenous caffeic acid inhibits the growth and enhances the lignification of the roots of soybean (Glycine max)
J. Plant Physiol.
(2011)
J. Deng et al.
Postharvest oxalic acid treatment induces resistance against pink rot by priming in muskmelon (Cucumis melo L.) fruit
Postharvest Biol. Technol.
(2015)
J.L. Ferrer et al.
Structure and function of enzymes involved in the biosynthesis of phenylpropanoids
Plant Physiol. Biochem.
(2008)
C.J. French et al.
An inactivator of phenylalanine ammonia-lyase from gherkin hypocotyls
Phytochemistry
(1975)
M.A. Gatto et al.
Phenolic extracts from wild edible plants to control postharvest diseases of sweet cherry fruit
Postharvest Biol. Technol.
(2016)
J.R. Gorny et al.
Quality changes in fresh-cut pear slices as affected by controlled atmospheres and chemical preservatives
Postharvest Biol. Technol.
(2002)
M. Hu et al.
Reduction of postharvest anthracnose and enhancement of disease resistance in ripening mango fruit by nitric oxide treatment
Postharvest Biol. Technol.
(2014)
M. Kalinowska et al.
Apples: content of phenolic compounds vs. variety, part of apple and cultivation model, extraction of phenolic compounds, biological properties
Plant Physiol. Biochem.
(2014)

V.N. Lima et al.

Antimicrobial and enhancement of the antibiotic activity by phenolic compounds: gallic acid, caffeic acid and pyrogallol

Microb. Pathog.

(2016)

Y. Liu et al.

Effect of postharvest acibenzolar-S-methyl dipping on phenylpropanoid pathway metabolism in muskmelon (Cucumis melo L.) fruits

Sci. Hortic.

(2014)

C. Pane et al.

Control of alternaria post-harvest infections on cherry tomato fruits by wild pepper phenolic-rich extracts

Crop. Prot.

(2016)

M. Prakash et al.

Biological functions of epicatechin: plant cell to human cell health

J. Funct. Foods

(2019)

B. Suárez et al.

Phenolic profiles, antioxidant activity and in vitro antiviral properties of apple pomace

Food Chem.

(2010)

S. Takshak et al.

Secondary metabolites and phenylpropanoid pathway enzymes as influenced under supplemental ultraviolet-B radiation in Withania somnifera Dunal, an indigenous medicinal plant

J. Photochem. Photobiol. B

(2014)

L.A. Terry et al.

Preformed antifungal compounds in strawberry fruit and flower tissues

Postharvest Biol. Technol.

(2004)

R.K. Toor et al.

Antioxidant activity in different fractions of tomatoes

Food Res. Int.

(2005)

D. Xu et al.

Fulvic acid-induced disease resistance to Botrytis cinerea in table grapes may be mediated by regulating phenylpropanoid metabolism

Food Chem.

(2019)

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Exogenous caffeic acid and epicatechin enhance resistance against Botrytis cinerea through activation of the phenylpropanoid pathway in apples

Highlights

Abstract

Introduction

Section snippets

Fruit material and fungal pathogen

Effect of two free phenols on incidence and lesion diameter

Discussion

Conclusion

CRediT authorship contribution statement

Declaration of Competing Interest

Acknowledgment

Phytochemistry

Plant Physiol. Biochem.

J. Plant Physiol.

Postharvest Biol. Technol.

Plant Physiol. Biochem.

Phytochemistry

Postharvest Biol. Technol.

Postharvest Biol. Technol.

Postharvest Biol. Technol.

Plant Physiol. Biochem.

Microb. Pathog.

Sci. Hortic.

Crop. Prot.

J. Funct. Foods

Food Chem.

J. Photochem. Photobiol. B

Postharvest Biol. Technol.

Food Res. Int.

Food Chem.