Buckwheat Antifungal Protein with Biocontrol Potential To Inhibit

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Cite This: J. Agric. Food Chem. 2019, 67, 6748−6756

Buckwheat Antifungal Protein with Biocontrol Potential To Inhibit Fungal (Botrytis cinerea) Infection of Cherry Tomato Caicheng Wang,†,‡,§,∥ Susu Yuan,†,‡,§,∥ Weiwei Zhang,†,‡,§ Tzibun Ng,⊥ and Xiujuan Ye*,†,‡,§ State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, ‡Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, and §Fujian Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China ⊥ School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China Downloaded via BUFFALO STATE on July 21, 2019 at 23:02:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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S Supporting Information *

ABSTRACT: A 11 kDa antifungal protein FEAP was purified from buckwheat (Fagopyrum esculentum) seed extract with a procedure involving (NH4)2SO4 precipitation and chromatography on SP-Sepharose, Affi-gel blue gel, Mono S, and Superdex peptide. Its N-terminal sequence was AQXGAQGGGAT, resembling those of buckwheat peptides Fα-AMP1 and Fα-AMP2. FEAP exhibited thermostability (20−100 °C) and acid resistance (pH 1−5). Its antifungal activity was retained in the presence of 10−150 mmol/L of K+, Mn2+, or Fe3+ ions, 10−50 mmol/L of Ca2+ or Mg2+ ions, and 50% methanol, 50% ethanol, 50% isopropanol, or 50% chloroform. Its half-maximal inhibitory concentrations toward spore germination and mycelial growth in Botrytis cinerea were 79.9 and 236.7 μg/mL, respectively. Its antifungal activity was superior to the fungicide cymoxanil mancozeb (248.1 μg/mL). FEAP prevented B. cinerea from infecting excised leaves, intact leaves, and isolated fruits of cherry tomato. Its mechanism involved induction of an increase in cell membrane permeability and a decrease in mitochondrial membrane potential. KEYWORDS: antifungal protein, purification, plant disease control, fruit preservation, mode of action

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Antifungal proteins and peptides, which represent the first line of defense to protect organisms from microbial attack, exist in animals, plants, and microorganisms.14,15 They demonstrate certain application potential in agriculture due to their considerable antifungal activity, safety, and less fungicide resistance.16,17 Currently, antifungal proteins and peptides have achieved some success in disease control, grain preservation, and improvement of plant variety.18−22 In this study, we purified and characterized a protein with inhibitory effect toward B. cinerea from buckwheat seeds and conducted an investigation on its control effect and mechanism of action. Our results indicated that the protein was new. It exhibited repressive activities toward spore germination and mycelial growth in B. cinerea and effectively protected cherry tomato from infection by B. cinerea. Through this study, we hope to lay a foundation for broadening the application of buckwheat antifungal protein and provide a stronger theoretical basis for the antifungal protein in the control of B. cinerea.

INTRODUCTION Botrytis cinerea is a necrotrophic fungal pathogen that occurs in any place where host plants grow, whether it be a tropical, subtropical, or cold temperate region. It infects at least 586 genera of plants including many common crops such as cucumber, strawberry, tomato, potato, and grape,1 causing gray mold or other illnesses. B. cinerea infects crops during the various growth periods of crops, but it usually enters host tissues at the early stages of crop development and lurks for a considerable period of time before the advent of a favorable environment and physiological changes in the host. Therefore, not only lesions of stems and leaves and rots of flowers and fruits are produced during the growth of the crop but also the seemingly healthy fruit and vegetable products are seriously damaged during transportation and storage.2,3 As the second important fungal pathogen in agriculture, B. cinerea has caused huge economic losses in agricultural production, and it has also incurred an enormous amount of expenditures for its disease management every year.3 At present, the disease control of B. cinerea mainly depends on chemical control,4 but the use of chemical agents is faced with many challenges today. The occurrence of pathogen resistance reduces the control effect of fungicides,5,6 and the residues of fungicides also brings safety risks to food and the environment.7,8 In view of chemical protection facing a series of issues, other methods of disease control have emerged. In recent years, biological control and the use of biological control agents have been developed for the prevention and control of B. cinerea.9−11 The biological control agents with antifungal activity include oils, phytohormones, antifungal proteins, antifungal peptides, and so on.11−13 © 2019 American Chemical Society

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MATERIALS AND METHODS

Materials. Buckwheat seeds were purchased from a local seed market. The fungicides carbendazim thiram bromothalonil (20% carbendazim, 10% thiram, 10% bromothalonil) and cymoxanil mancozeb (8% cymoxanil, 64% mancozeb) were purchased from a local pesticide store. The Affi-gel blue gel used for chromatography was purchased from Bio-Rad Laboratories, and SP-Sepharose, Mono S Received: Revised: Accepted: Published: 6748

February 18, 2019 May 19, 2019 May 28, 2019 May 28, 2019 DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756

Article

Journal of Agricultural and Food Chemistry 5/50 GL column, and Superdex peptide 10/300 GL column were purchased from GE Healthcare. B. cinerea used in the experiments was provided by Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University. Purification of Antifungal Protein. Buckwheat seeds (190 g) were added to 1 L of 20 mmol/L NH4OAc buffer (pH 4.6), soaked overnight, and then homogenized. The homogenate was centrifuged (10 000 × g, 4 °C, 30 min). The supernatant was retained as the crude protein extract and then precooled at 0 °C. Ammonium sulfate was added to the precooled extract to achieve a saturation of 50%, and the extract was centrifuged (10 000 × g, 4 °C, 30 min) after standing for 4 h. The supernatant was retained, and ammonium sulfate was again added to bring the saturation to 90%. After standing for 4 h, the mixture was centrifuged, and the supernatant was discarded to obtain an active protein precipitate. The protein precipitate was redissolved in 20 mmol/L NH4OAc buffer (pH 4.6) and applied to a cationexchange SP-Sepharose column (5 cm × 16 cm). After the unadsorbed proteins had been washed off with 20 mmol/L NH4OAc buffer (pH 4.6), the adsorbed proteins were eluted successively with NH4OAc buffer containing 0.2, 0.5, and 1 mol/L NaCl. The chromatographic fraction with antifungal activity was dialyzed against double-distilled water in a dialysis bag with a molecular weight cutoff of 3500 Da and then freeze dried into dry powder. The dry powder was redissolved in 20 mmol/L Tris-HCl buffer (pH 7.5) and applied to an affinity column filled with Affi-gel blue gel. After the unadsorbed proteins had been washed off with 20 mmol/L Tris-HCl buffer (pH 7.5), the adsorbed proteins were eluted with 20 mmol/L Tris-HCl buffer (pH 7.5) containing 1 mol/L NaCl. The chromatographic fraction with antifungal activity was dialyzed and lyophilized, redissolved in 20 mmol/L NH4OAc buffer (pH 4.6), and then loaded onto a Mono S 5/50 GL column attached to the AKTA purifier system (GE Healthcare) for cation-exchange chromatography using fast protein liquid chromatography (FPLC). The adsorbed proteins were eluted with a gradient of 0−0.16 mol/L NaCl and 1 mol/L NaCl in NH4OAc buffer, and the active component was dialyzed and lyophilized. The protein powder was redissolved in 20 mmol/L of NH4OAc buffer (pH 4.6) and subjected to chromatography on a Superdex peptide 10/300 GL column attached to the AKTA purifier system for gel filtration chromatography, and the proteins were eluted with the same buffer. In each chromatographic step, the antifungal activity of each chromatographic fraction was determined by the following filter paper method. Activated B. cinerea was inoculated to a PDA plate and cultured at 28 °C. When the diameter of the colony reached 3−5 cm, sterile filter papers with a diameter of 6 mm were placed 5 mm from the edge of the colony. A 20 μL aliquot of each eluate was added to the filter paper, and buffer (20 μL) was added as a negative control. The plates were further incubated at 28 °C, and the growth of hyphae at the edge of the colony was observed within 1−2 days.23 The protein concentration of each chromatographic fraction with antifungal activity was determined by the BCA method using a kit, and then the yield was calculated. Molecular Weight Determination of Antifungal Protein by Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. The molecular weight of the antifungal protein was determined by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE)24 employing 4% stacking gel, 10% spacer gel, and 16.5% separating gel, and Coomassie brilliant blue dye was used for gel staining at the end of electrophoresis. Qualitative Identification of Antifungal Protein and Determination of N-Terminal Amino Acid Sequence. The antifungal protein samples were electrophoresed as described above, and the gel used for transfer had previously been electrophoresed at 5 mA for 2 h before loading of the sample. After electrophoresis the gel was stained with Coomassie brilliant blue, and the antifungal protein band was excised after destaining of the gel to remove excess stain. Another gel was taken for transferring the sample to a PVDF membrane, followed by staining with Ponceau dye, and the residue was then washed off. The above two samples were sent to Shanghai Applied Protein Technology Co., Ltd. for identification and also for

determination of N-terminal amino acid sequence. Identification of antifungal protein was performed using matrix-assisted laser desorption/ionization with time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), and the information regarding the resulting peptide fragments was searched by using Mascot 2.2 software to collect qualitative information about the antifungal protein. The N-terminal amino acid sequence of the antifungal protein was determined using the Edman degradation method, and the sequence obtained was aligned using the Blast method in the nr database of the NCIB database to gather information on proteins similar to the antifungal protein. Determination of Stability of Antifungal Protein Based on Changes in Activity. The effects of temperature, acid−base, metal ions, and organic solvents on the activity of the antifungal protein were assayed by the filter paper method mentioned above after the antifungal protein had been subjected to different treatments. In the thermostability experiment, 100 μL aliquots of the solution of the antifungal protein were separately exposed to 20−120 °C for 30 min. Thermal treatments at 0−100 °C were performed by using a water bath, whereas treatments at 120 °C were conducted by using a steam pot. In the acid−base stability experiment, 10 μL aliquots of the solution of the antifungal protein were separately mixed with 90 μL of PBS buffer (pH 1−14) and left at room temperature for 2 h. In the metal ion stability experiment, 50 μL aliquots of the solution of the antifungal protein were separately mixed with 50 μL of a solution containing KCl, CaCl2, MgCl2, MnCl2, or FeCl3 at a concentration of 10−150 mmol/L and placed at room temperature for 2 h. In the organic solvent stability test, aliquots of the solution of the antifungal protein were separately mixed with 50 μL of methanol, ethanol, isopropanol, or chloroform and allowed to stand at room temperature for 2 h. In the above experiments, the untreated solution (pH 7) of the antifungal protein was used as the positive control for comparison with the treated solution of the antifungal protein. The final concentration of antifungal protein in all treated samples and positive control was 0.5 mg/mL. In the thermostability and acid−base stability experiments, PBS buffer (pH 7) was used as the negative control. In the metal ion stability experiment, solutions of KCl, CaCl2, MgCl2, MnCl2, and FeCl3 at a concentration of 150 mmol/L were used as the negative control. In the organic solvent stability experiment, solutions of methanol, ethanol, isopropanol, and chloroform in water at the concentration of 50% (v/v) were used as the negative control. Determination of Antifungal Activity of Antifungal Protein. The half-maximal inhibitory concentrations (IC50) of the antifungal protein toward spore germination and hyphal growth in B. cinerea were determined. The method for determining IC50 toward spore germination was as follows. B. cinerea was inoculated on PDA medium and cultured for 14 days at 28 °C in the dark. The fungal suspension was obtained by adding 5 mL of sterile water to the plate, followed by scraping the surface of the colony with an applicator, and the spore suspension was obtained by filtering the fungal suspension through three layers of lens paper twice to remove the hyphae. The spore concentration of the suspension was adjusted to 1 × 107 /mL and mixed with an equal volume of 0.05% glucose solution, and then the mixture was mixed with an equal volume of a sterile solution of the antifungal protein at final concentrations of 23.2, 46.4, 92.8, 185.6, 371.2, and 742.4 μg/ mL. A 20 μL aliquot of the final mixture was added to the center of the concave slide which was placed in a Petri dish containing two layers of sterile wet filter paper and then incubated at 28 °C for 10 h. In the control group, the solution of the antifungal protein was replaced with PBS buffer (pH 7), and there were 3 replicates in each of the control group and the treatment group. In each group, at least 100 spores were observed under an optical microscope and the number of germinated spores was recorded. The rate of inhibition of germination of B. cinerea spores by the antifungal protein was calculated by the following formula: germination inhibition rate = (spore germination rate of control group − spore germination rate of treatment group)/spore germination rate of control group × 100%, and the IC50 value was calculated by employing the SPSS software. 6749

DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756

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Journal of Agricultural and Food Chemistry

Figure 1. Elution profiles of antifungal protein FEAP: (A) cation-exchange chromatography of proteins precipitated by 50%−90% ammonium sulfate on SP-Sepharose column; (B) affinity chromatography of component SP2 on Affi-gel blue gel column; (C) cation-exchange chromatography of component B3 on Mono S column; (D) gel filtration chromatography of component M4 on Superdex Peptide column. in a water bath for 3 min, followed by decolorizing with chloral overnight, and then placed in a 50% glycerol stock solution. The circular leaves were placed on the slides with the back side facing up, and the growth status of the pathogenic fungus in the leaves was observed under a light microscope. In order to demonstrate the effect of antifungal protein on the biocontrol of B. cinerea, integrated leaves and excised cherry tomato fruits were used to conduct the following experiment. Spore suspensions of B. cinerea at a final concentration of 1 × 107 spores/ mL containing 2.30 or 4.59 mg/mL of the antifungal protein were prepared separately. Two groups of leaves of 1-month-old cherry tomato seedlings were punctured with a needle, followed by inoculation with the above two spore suspensions in a volume of 10 μL. In the negative control group, the spore suspension containing antifungal protein was replaced with a spore suspension (1 × 107 spores/mL) without antifungal protein, and in the blank control group, spore suspension was replaced with sterile water. In each group, three samples were set. After the seedlings had continued to grow for 9 days, the leaves of each group were photographed by utilizing a camera at the same object distance, and the lesion area of leaves was calculated by the Photoshop software.26 Cherry tomato fruits were soaked in 75% alcohol for 5 s and then rinsed twice with sterile distilled water, followed by puncturing with a sterile needle. Two groups of above fruits were inoculated with the two aforementioned spore suspensions containing antifungal protein in a volume of 10 μL. Spore suspension (1 × 107 spores/mL) without antifungal protein and sterile water were used instead of antifungal protein to treat with the same method as negative control and blank control. In each group, six samples were set. After the inoculated fruits had been placed at 28 °C for 8 days, the wound diameter of each group of fruits was measured. For the experimental data, the significant analysis was performed with the Student t test. Study of Mechanism of Action of Antifungal Protein. The changes of cell membrane permeability and mitochondrial membrane potential were observed after staining B. cinerea hyphae with the fluorescent dyes SYTOX green and Rhodamine 123.27,28 SYTOX green is a nucleic acid dye that can enter cells when the plasma membrane is damaged. Rhodamine 123 is a cell membranepenetrating fluorescent dye that can enter the mitochondrial matrix

The method for determining the IC50 toward mycelial growth was as follows. A 1200 μL amount of PDA medium (0.7% agar) cooled to 40−50 °C was mixed with 300 μL of a sterile solution of the antifungal protein at final concentrations of 33.3, 100, 300, and 900 μg/mL and then added to the culture dish (30 mm × 15 mm). After the medium had solidified, the hyphal disk of B. cinerea with a diameter of 6 mm, which was severed from the edge of the colony by using a sterile puncher, was inoculated at the center of the dish, and each plate was cultured at 28 °C until the colony in the negative control group had grown to the edge of the dish. In the negative control group, the solution of antifungal protein was replaced by PBS buffer (pH 7). In the positive control group, the solution of antifungal protein was replaced by the fungicides carbendazim thiram bromothalonil and cymoxanil mancozeb. There were 3 replicates in each of the control group and the treatment group. After measuring the colony diameter of B. cinerea by the cross method, the rate of inhibition of hyphal growth of the antifungal protein against B. cinerea was calculated by the following formula: growth inhibition rate = (colony area of control groupcolony area of treatment group)/ colony area of control group) × 100%, and the IC50 was calculated by utilizing the SPSS software. Biocontrol Effect of Antifungal Protein. In order to assay the infectivity of B. cinerea on cherry tomato and the effect of antifungal protein on the growth of the pathogen in cherry tomato tissue, detached cherry tomato leaves were used to carry out the following experiment. The leaves of 1-month-old cherry tomato seedlings were soaked in 75% ethanol for 5 s and then washed twice with sterile distilled water. After the leaves had dried naturally, they were cut into circular disks with a diameter of 1.2 cm using a sterile punch. The circular disks were immersed in sterile water and a solution of the antifungal protein at a concentration of 4.59 mg/mL for 5 min and then placed in a Petri dish containing two layers of filter paper after the liquid on the leaves had dried. A 10 μL aliquot of a B. cinerea spore suspension at a concentration of 1 × 105 /mL was added to the center of the circular disks immersed in sterile water and antifungal protein solution, and the Petri dish was then incubated at 28 °C. After 2 days of culture, the circular leaves of the two groups with different treatments were immersed in alcoholic lactophenol trypan blue in a 100 °C water bath for 10 min,25 transferred to alcoholic lactophenol 6750

DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756

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Journal of Agricultural and Food Chemistry in normal cells and is used as a probe of the mitochondrial transmembrane potential. When it enters the mitochondrial matrix, its fluorescence intensity dwindles or fades away. The well-developed mycelium of B. cinerea cultured on a PDA plate was inoculated into PDA liquid medium or PDA liquid medium containing the antifungal protein at the concentration of 0.2 mg/mL, followed by culture at 28 °C with shaking (150 rpm/min). After 1 day of culture, SYTOX green at a final concentration of 5 μmol/L or Rhodamine 123 at a final concentration of 4 μg/mL was separately added to the fungal suspension and then allowed to stand in the dark for 30 min. The hyphae were collected and washed with PBS buffer to prepare a smear. The staining of hyphae was observed under a laser scanning confocal microscope. The excitation and emission wavelengths used for observation of SYTOX green staining were 488 and 525 nm, and the excitation and emission wavelengths used for observation of Rhodamine 123 staining were 561 and 595 nm.

cation-exchange chromatography of the dissolved precipitate (derived from the crude protein extract by using 50%−90% ammonium sulfate) on an SP-Sepharose column (Figure 1A). The active chromatographic fraction B3 was subsequently derived from SP2 by affinity chromatography on an Affi-gel blue gel column (Figure 1B). Cation-exchange chromatography of B3 on Mono S yielded the active chromatographic fraction M4 (Figure 1C). Finally, the active chromatographic fraction SU2 was obtained from M4 by gel filtration chromatography on a Superdexpeptide column (Figure 1D). The yield of the active fraction at each chromatographic step is listed in Table 1. The chromatographic fraction SU2 exhibited a single protein band with a molecular weight of 11 kDa in Tricine-SDS-PAGE (Figure 2), which was named FEAP. FEAP showed a reliable matching score with the buckwheat peptides Fα-AMP1 and Fα-AMP2 by MALDI-TOF/TOF MS analysis, and it also manifested the highest similarity to Fα-AMP1 and Fα-AMP2 by N-terminal sequence alignment. The N-terminal sequence of FEAP was AQXGAQGGGAT (X corresponds to an unknown amino acid), and its similarity with both of the above two buckwheat peptides was 90.91%. The results of mass spectrometric identification and amino acid sequence alignment of FEAP are shown in Tables 2 and 3, respectively. The stability study disclosed that FEAP displayed pronounced thermostability and stability in ambient acidic pH. Its antifungal activity did not undergo a significant decline after exposure to 20−100 °C for 30 min and following incubation in acidic solutions of pH 1−5 for 2 h, but its activity completely vanished after heat treatment at 120 °C and after treatment with alkaline solutions of pH 10−14. FEAP also showed favorable stability when confronted with organic solvents: its antifungal activity was retained in the presence of methanol, ethanol, isopropanol, or chloroform at a concentration of 50%. The activity of FEAP showed a certain difference in the presence of different metal ions. Its antifungal activity remained untarnished in the presence of K+, Mn2+, and Fe3+ ions at a concentration of 10−150 mmol/L and Ca2+ and Mg2+ ions at a concentration of 10 and 50 mmol/L, respectively, but its antifungal activity was attenuated in the presence of Ca2+ and Mg2+ ions at 100 mmol/L and higher concentrations. When the concentration of FEAP was 185.6, 371.2, and 742.4 μg/mL, spore germination in B. cinerea was inhibited by 65%, 75%, and 91%, respectively (Figures 3A and 4A). When the concentration of FEAP was 300 and 900 μg/ mL, mycelial growth in B. cinerea was inhibited by 54% and 91%, respectively (Figures 3B and 4B). The IC50 values of FEAP in spore germination and mycelial growth in B. cinerea as calculated by the SPSS software were 79.9 and 236.7 μg/mL, respectively. For comparison, the IC50 values of fungicides including carbendazim thiram bromothalonil and cymoxanil

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RESULTS AND DISCUSSION A highly purified antifungal protein was obtained from the crude protein extract of buckwheat seeds by employing a Table 1. Yields of Active Components in Different Chromatographic Steps purification stage

fraction

yield (mg)

ammonium sulfate precipitation cation-exchange chromatography affinity chromatography cation-exchange chromatography gel filtration chromatography

50−90% NH4SO4 SP2 B3 M4 SU2

1184 418 303 26.9 1.1

Figure 2. Molecular weight determination of FEAP by Tricine-SDSPAGE.

protocol that entailed ammonium sulfate precipitation, cationexchange chromatography, affinity chromatography, cationexchange chromatography,and gel filtration chromatography. The active chromatographic fraction SP2 was obtained by

Table 2. MALDI-TOF/TOF MS Results of Peptide Fragments Derived from FEAP protein species matched fragment protein MW sequence IDa protein score C.I. %b

Fa-AMP1

Fa-AMP2

Fagopyrum esculentum YCGAGCQSNCK AQCGAQGGGATCPGGLCCSQWGWCGSTPK 3879.1 Da P0DKH7.1 100

F. esculentum YCGAGCQSNCR AQCGAQGGGATCPGGLCCSQWGWCGSTPK 3906.1 Da P0DKH8.1 99.5

a

According to the Sequence ID, more information on the peptides can be obtained from the protein database of the NCIB database. bIt represents the matching degree of FEAP with Fa-AMP1 and Fa-AMP1, and the value >95 indicates that the result is reliable. 6751

DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756

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F. esculentum AQCGAQGGGATCPGGLCCSQWGWCGSTPKYCGAGCQSNCR 3906.1 Da P0DKH8.1 91% F. esculentum AQCGAQGGGATCPGGLCCSQWGWCGSTPKYCGAGCQSNCK 3879.1 Da P0DKH7.1 91% F. esculentum AQXGAQGGGAT (N-terminal sequence) 11 kDa species sequence protein MW sequence IDa identitiesa

a According to the Sequence ID, more information on the peptides can be obtained from the protein database of the NCIB database. bIt indicates the similarity of amino acid sequence of FEAP compared with Fa-AMP1 and Fa-AMP2.

Fa-AMP2 Fa-AMP1 FEAP protein

Table 3. Alignment Results of the N-Terminal Amino Acid Sequence of FEAP

Journal of Agricultural and Food Chemistry

Figure 3. Inhibition of spore germination and mycelial growth in B. cinerea by FEAP: (A) inhibition rate of spore germination; (B) inhibition rate of mycelial growth; Data are presented as mean with SEM.

mancozeb on the mycelial growth of B. cinerea were 2.4 and 248.1 μg/mL, respectively. The excised cherry tomato leaves infected with B. cinerea were observed under the microscope. It was found that B. cinerea spores on leaves soaked in sterile water underwent normal germination, invaded the leaves through the stomata (Figure 5A), and grew extensively inside the leaf tissue (Figure 5B). On the contrary, B. cinerea spores on leaves soaked in a solution of the antifungal protein failed to germinate (Figure 5C), indicating that FEAP effectively forestalled B. cinerea infection of excised cherry tomato leaves. When intact cherry tomato leaves were infected with B. cinerea, leaves merely inoculated with B. cinerea spores were severely affected, with lesions spread throughout the leaves (the mean area of lesion was defined as 100%). Leaves inoculated with spore suspension containing 2.30 mg/mL of FEAP were also affected, and the mean area of lesion was reduced to 64% (Figure 6A). However, when the concentration of FEAP in the spore suspension was raised to 4.59 mg/mL, the average lesions area in the inoculated leaves was further reduced to 2.3%. Some of the leaves were not affected and had a similar appearance to the blank control group (Figure 7A). B. cinerea infection caused serious epidermal invagination and tissue rot in the isolated cherry tomato fruit. In the negative control, the average diameter of the wound in the fruit was 9.1 mm. When the fruits were inoculated with a spore suspension containing FEAP, the severity of the disease in the fruits decreased as the FEAP concentration increased (Figure 6B). At the FEAP concentration of 2.30 mg/mL, the average diameter of the wound in the fruits was reduced to 4.8 mm. At the FEAP 6752

DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756

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Journal of Agricultural and Food Chemistry

Figure 4. Inhibitory effects of FEAP toward spore germination and mycelial growth in B. cinerea: (A) spore germination status; and (B) mycelial growth status; numerical value indicates the concentration (μg/mL) of FEAP in the medium.

(Figure 7B). The aforementioned results revealed that FEAP can effectively thwart B. cinerea infection on integrated leaves and excised cherry tomato fruits. Fluorescence staining experiments revealed that after staining with SYTOX green and Rhodamine 123 fluorescence appeared in the hyphae of the FEAP-treated group, while fluorescence was indiscernible in the hyphae of the control group (Figures 8 and 9), indicating that FEAP elicited an increase in cell membrane permeability together with a decline in mitochondrial membrane potential. In this study, we purified an antifungal protein FEAP from the crude extract of F. esculentum seeds by using a protocol that comprised ammonium sulfate precipitation and simple chromatographic procedure. The methodology is routine and facile. We obtained 1.1 mg of antifungal protein from 190 g of seeds. It has been reported that a protein and several peptides with antifungal activity have been isolated from Fagopyrum spp., such as protein FtTI, peptides Fα-AMP1 and Fα-AMP2, and another peptide.29−31 Compared with them, the yield of FEAP (0.58 mg per 100 g seeds) was similar to that of FαAMP1 (0.6 mg per 100 g seeds) but slightly higher than that of Fα-AMP2 (0.47 mg per 100 g of seeds). In terms of molecular mass or protein type, FEAP also has some distinctive characteristics compared with Fα-AMP1 and Fα-AMP2. The molecular weight of FEAP was 11 kDa, while those of FαAMP1, Fα-AMP2, and another antifungal peptide were 3879, 3906, and 3900 Da,29,30 respectively. FtTI exhibited a molecular mass of 14 kDa when analyzed by SDS-PAGE and showed two isoforms (11 487 and 13 838 Da) as analyzed by MALDI-TOF,31 which was closer to that of FEAP. However, the results of mass spectrometric identification and N-terminal sequence alignment show that FEAP is structurally similar to Fα-AMP1 and Fα-AMP2, which belong to the defensin family according to Fujimura et al.29 while FtTI is a trypsin inhibitor. Some peptide fragments derived from FEAP match the defensin peptide, thus, we compared FEAP with defensin-like peptides and antifungal proteins from plants. The antifungal activity of FEAP is stable under acidic conditions. Although it has less remarkable pH stability than several defensin-like peptides isolated from buckwheat and other plants,30,32,33 it has certain advantages in thermal stability. It remains stable

Figure 5. Growth status of B. cinerea in excised cherry tomato leaves: (A, B) leaves were soaked in sterile water; (C) leaves were soaked in FEAP solution.

concentration of 4.59 mg/mL, the average diameter of the wound was further reduced to 2.9 mm, and the fruit epidermis at the inoculation site in some fruits was basically smooth 6753

DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756

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Journal of Agricultural and Food Chemistry

Figure 6. Incidence of infection in cherry tomato: (A) relative lesion area in intact leaves; (B) wound diameter of excised fruit; (*) significance at P < 0.05; Data are presented as mean with SEM, numerical value indicates the concentration (μg/mL) of FEAP.

Figure 7. Effects of FEAP on prevention of B. cinerea infection of cherry tomato: (A) prevention effect in intact leaves; (B) prevention effect in excised fruit; numerical value indicates the concentration (μg/mL) of FEAP.

Figure 8. FEAP elicited an increase in cell membrane permeability of B. cinerea observed by SYTOX green staining.

Figure 9. FEAP elicited a decrease in mitochondrial membrane potential of B. cinerea observed by Rhodamine 123 staining.

following heat treatment at 20−100 °C, which is consistent with the findings on the defensin-like peptide NRBAP.33 It is superior in thermostability to defensin-like antifungal peptides

from buckwheat (0−70 °C) and brown kidney beans (20−80 °C) and antifungal proteins from cabbage (0−65 °C) and banana (20−50 °C).30,32,34,35 Generally, defensins are rich in 6754

DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756

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Journal of Agricultural and Food Chemistry ORCID

disulfides and convergently utilize double-stranded or triplestranded beta-sheets cross-linking a disulfide network into a tight core. Disulfide bonding endows defensins with high stability to temperature,36 and the excellent thermostability of FEAP may be associated with this. In addition to its remarkable thermostability, FEAP also has tolerance to organic solvents. FEAP brings about a rise in membrane permeability and a fall in mitochondrial membrane potential. The former is commonly observed in several plant defensin-like peptides and other plant antifungal proteins,33−35,37,38 and the latter is similar to a mutant Allium sativum leaf agglutinin (mASAL).27 FEAP is isolated from F. esculentum. Compared with the reports of Fujimura et al. and Leung and Ng,29,30 the present investigation has made further progress in research on the antifungal activity of FEAP and addresses more regarding its possible practical application. FEAP not only evinces pronounced stability and demonstrates multiple modes of action but also its antifungal activity is characterized by certain advantageous features. Its IC50 value toward hyphal growth in B. cinerea is lower than the fungicide cymoxanil mancozeb. It is efficacious in preventing B. cinerea infection on cherry tomato. The data signify that FEAP has a promising biocontrol potential. Although FEAP exhibits certain potential for application, research on FEAP has yet to be expanded. Mechanistically, FEAP may suppress fungal growth by upregulating cell membrane permeability and disrupting mitochondrial membrane potential. Cell membrane permeabilization may involve binding to the cell wall,39 and the decrease in mitochondrial potential may further involve apoptosis of fungal cells.40 Some proteins have been reported to cause apoptosis in fungi, such as mASAL protein, which leads to loss of mitochondrial membrane potential and accumulation of intracellular reactive oxygen species in Rhizoctonia solani, and induces programmed cell death of mycelial cells.27 The TUBP1 protein isolated from Bacillus axarquiensi brings about mitochondria-mediated apoptotic cell death in Verticillium dahliae, involving a reduction in enzyme activity (mitochondrial dehydrogenases, F0F1-ATPase, malate dehydrogenase, and succinate dehydrogenase), an increase in reactive oxygen species, a decrease in mitochondrial membrane potential, release of cytochrome c, and activation of metacaspase.41 Whether FEAP also has the above-mentioned effects remains to be verified. In terms of application, it has been reported that the combination of antifungal peptide and chemical fungicide greatly improves the antifungal effect,42 and the expression of antifungal protein in plants increases the resistance to pathogenic fungi.43 In addition, favorable progress has been made in the expression and production of recombinant antifungal proteins.44,45 All of the studies mentioned above are feasible directions for further research on the applications of FEAP.

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Xiujuan Ye: 0000-0002-0231-0374 Author Contributions ∥

C.W. and S.Y.: These authors contributed equally to this work.

Funding

This study was supported by the University-Industry Cooperation Project of Fujian Provincial Department of Science and Technology (2018N5005). Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01144.

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REFERENCES

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Tricine-SDS-PAGE gel showing purification of FEAP (PDF)

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DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756

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DOI: 10.1021/acs.jafc.9b01144 J. Agric. Food Chem. 2019, 67, 6748−6756