New Nematotoxic Indoloditerpenoid Produced by Gymnoascus reessii

Mar 25, 2017 - Many fungi produce metabolites antagonistic or even lethal to nematodes, and such compounds offer great promise for the development of ...
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New Nematotoxic Indoloditerpenoid Produced by Gymnoascus reessii za-130 Ting Liu,† Susan L. F. Meyer,‡ David J. Chitwood,*,‡ Kamlesh R. Chauhan,# Dan Dong,† TaoTao Zhang,† Jun Li,§ and Wei-cheng Liu† †

Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Science, Beijing 100097, China Mycology and Nematology Genetic Diversity and Biology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Building 010A, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States # Invasive Insect Biocontrol and Behavior Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Building 007, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States § Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China ‡

ABSTRACT: Chemical investigation of the fungal strain Gymnoascus reessii za-130, which was previously isolated from the rhizosphere of tomato plants infected by the root-knot nematode Meloidogyne incognita, led to the isolation and identification of a new indoloditerpenoid metabolite designated gymnoascole acetate. Its structure was established by spectroscopic methods including 1D- and 2D-NMR and MS analyses. Gymnoascole acetate demonstrated strong adverse effects on M. incognita secondstage juvenile (J2) viability; exposure to 36 μg/mL for 24 h induced 100% paralysis of J2 (EC50 = 47.5 μg/mL). Gymnoascole acetate suppressed M. incognita egg hatch relative to controls by >90% at 133 μg/mL after 7 days of exposure. The numbers of root galls and J2 in both soil and roots were significantly reduced (p = 0.05) by treatment with 2−200 μg/mL gymnoascole acetate/kg soil, compared to untreated control plants; nematode suppression increased with gymnoascole acetate concentration. This study demonstrated the nematotoxicity of gymnoascole acetate and indicates that it might be a potential biobased component in integrated management of M. incognita. KEYWORDS: fungus, gymnoascole acetate, indoloditerpenoid, Gymnoascus reessii, Meloidogyne incognita, nematicide, nematode, nematotoxin, terpenoid

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INTRODUCTION Plant-parasitic nematodes are responsible for more than U.S. $125 billion annually in worldwide crop losses.1 Among the most damaging phytoparasitic nematodes are the root-knot nematodes (RKN; Meloidogyne spp.),2 especially Meloidogyne incognita (Kofoid & White) Chitwood, which infects almost all agricultural plants.3 Several strategies with various degrees of success are used to control these nematodes, including cultural practices such as crop rotation, deployment of resistant plant varieties, and application of chemical nematicides or, to a lesser extent, biological control agents. Because of environmental and human health concerns, substantial regulatory action has been directed at the reduction in usage of specific synthetic nematicides.4 Continued crop yield losses demonstrate that there is an urgent need to develop alternative, environmentally friendly, safe, and effective management strategies for RKN.5 The search for alternatives, including biocontrol agents and natural compounds,6 has drawn great attention in recent years. Among biological control agents, fungi provide one of the most diverse and important microbial resources. Many fungi produce metabolites antagonistic or even lethal to nematodes, and such compounds offer great promise for the development of future chemical management tools.7−16 One widespread soil fungus is Gymnoascus reessii Baran., which produces numerous secondary metabolites including several aromatic butenolides with a myriad of biological effects, such as antifungal, antioxidative, cytoprotective, and antitumor activities.17,18 © 2017 American Chemical Society

However, the nematicidal activities of the compounds produced by this fungus have been rarely investigated. During a search for bioactive compounds against RKN, a strain of G. reessii (za130) obtained from soil samples infested with M. incognita from Beijing exhibited distinct activities against this nematode. In earlier studies on the secondary metabolites from this strain, a new antinematodal principle was identified,19 although the identification is believed to be incorrect (unpublished results). The purpose of the present study was to continue the search for nematotoxic compounds in G. reessii. Different cultural conditions were used to increase the likelihood of discovery of additional compounds active in hatching and motility bioassays using M. incognita. One new nematotoxic indoloditerpenoid (Figure 1), gymnoascole acetate, was isolated and identified via the procedures described herein.

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were determined on a Rudolph Autopol IV automatic polarimeter. IR spectra were recorded on a Thermo Nicolet (Madison, WI, USA) Nexus 470 FT-IR spectrophotometer with KBr pellets. The NMR spectra were obtained with Varian (Palo Alto, CA, USA) INOVA-500 spectrometers operating at 500 MHz for 1H and at 125 MHz for 13C.

Received: Revised: Accepted: Published: 3127

September 29, 2016 March 13, 2017 March 25, 2017 March 25, 2017 DOI: 10.1021/acs.jafc.6b04353 J. Agric. Food Chem. 2017, 65, 3127−3132

Article

Journal of Agricultural and Food Chemistry

sieve. The extracted eggs were gently washed with tap water to remove NaOCl.22,23 Two bioassays were conducted. For the J2 motility assay, gymnoascole acetate was dissolved in DMSO and diluted with distilled water to concentrations of 10, 20, 50, 100, 200, and 400 μg/ mL to assay for nematotoxic activity. Aliquots (0.9 mL) were transferred to wells of 24-well tissue culture plates, and 100 μL of water containing 100 J2s was added to each well, thereby containing treatment solutions ranging from 9.0 to 360 mL. Each treatment was replicated three times with distilled water as a control; this experiment and all other bioassays were repeated. Immotile and motile nematodes were counted after 12, 24, and 36 h. The nematodes were considered to be immotile if they did not move when prodded with a fine needle. Mean percentage inhibition of motility (i.e., nematotoxicity) was calculated.15 For an egg hatch bioassay, 1.0 mL of egg suspension (containing about 250 eggs/mL) was combined with 2 mL of test solutions prepared at concentrations of 10, 20, 50, 100, 200, and 400 μg/mL in 6 cm diameter plastic Petri dishes, thereby creating final concentrations of 6.7, 13.3, 33.3, 66.7, 133.3, and 267 μg/mL. Eggs in sterilized distilled water served as controls; there were four replicates of all treatments and controls in each of two trials. Plate lids were sealed with Parafilm, and the plates were kept at 25 °C. After 7 days, hatched J2s were counted with the use of an inverted microscope, and hatch suppression percentage was calculated.15 Effects of Gymnoascole Acetate on Nematode Reproduction on Tomato Plants in Greenhouse Pots. Tomato seedlings with three leaves (4 weeks old) were transplanted individually into 6 cm diameter plastic pots filled with steam-sterilized sandy loam soil amended with organic peat and sand (field soil/organic peat/sand = 1:3:4). Immediately following transplant, an aqueous suspension of 1000 J2s of M. incognita was then placed into three holes, each 3 cm deep, distributed evenly around each seedling. Ten milliliters of gymnoascole acetate solutions of concentrations of 2, 20, or 200 μg/ mL were then divided into the three holes per pot, and the holes were covered with soil. Sterilized distilled water was used as the control. A completely randomized design was used; each treatment was replicated 10 times, and plants were maintained under natural lighting and watered as needed. Thirty-five days after inoculation with M. incognita, the numbers of root galls were counted. Additionally, the M. incognita J2s in the roots and soil from the pot were stained or extracted, respectively, and then enumerated. For J2 enumeration, the soil from the entire pot was thoroughly mixed, a 200 g sample was removed, and nematodes were isolated with a centrifugation−sucrose flotation method.24 Root-knot nematodes inside infected roots were observed with acid fuchsin staining as previously described,25 with slight modification. Roots were placed in a 1.5% sodium hypochlorite solution for 4 min with occasional agitation, gently but extensively washed with running tap water, soaked in tap water for 15 min, transferred to the acid fuchsin staining solution, and heated to boiling for about 30 s. The samples were left to cool at room temperature, rinsed with running tap water, placed in glycerin acidified with a few drops of 5 N HCl, heated to boiling, and cooled. Statistical Analyses. All experiments utilized a completely randomized design and were repeated, except for the greenhouse pot experiment, which was performed once. Stat.10 for Windows (SPSS Inc. 2000: SPSS Base 10.1 User’s Guide: SPSS Inc., Chicago, IL, USA) was used for statistical analysis, including probit analysis for EC50 calculation. Duncan’s new multiple-range test was employed to test for significant differences among treatments at p = 0.05; means were compared with the least significant differences (LSD) test (p = 0.05).

Figure 1. Chemical structure of compound 1 (gymnoascole acetate). The HRESIMS data were determined on a Shimadzu (Kyoto, Japan) LCMS-IT-TOF system incorporating a Prominence UFLC system and an ESI interface. Silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), LiChroprep RP-C18 (40− 63 μm, Merck, Darmstadt, Germany), and Sephadex LH-20 (Pharmacia, Uppsala, Sweden) were used for open column chromatography. HPLC was performed on a Waters (Milford, MA, USA) system fitted with a model 2535 pump and a model 2998 photodiode array detector set at 228 nm. A semipreparative RP-HPLC column (Grace C18, 250 × 10 mm, 5 μm, Hesperia, CA, USA) attached to the same HPLC system was employed for isolation. The TLC was performed on silica gel GF254 plates developed with various ratios of diethyl ether/ethyl acetate (10:1−1:1); compounds were detected by spraying with vanillin reagent (1 g of vanillin in 100 mL of concentrated H2SO4) and heating at 110 °C until optimal color development occurred. The purified compound utilized in bioassays exceeded 95% purity as judged by HPLC and supported by 1H NMR analysis. Culture and Fermentation of G. reessii za-130. G. reessii za-130 was previously isolated from M. incognita-infested rhizosphere soil collected from a suburb of Beijing, China.20 A stock culture in 20% (v/ v) glycerol suspension was maintained at −20 °C and deposited in the Microbial Laboratory of the Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Science, Beijing, China. A slant culture was subcultured on corn meal agar for 7 days, and then 1 cm disks of growing colonies were cut from the margin of each plate, inoculated into 80 Erlenmeyer flasks containing solid rice medium (rice, 80 g; water, 120 mL), and cultured at 25 °C for 40 days to maximize the production of diverse metabolites. Extraction and Isolation. The combined mycelia plus culture filtrate from G. reessii za-130 cultures were extracted successively with 3.0 L of EtOAc three times at room temperature. The combined extracts were concentrated to dryness in vacuo to give the residue (17.73 g), which was subjected to silica gel column chromatography (200−300 mesh, Qingdao Marine Chemical Factory; 6 × 40 cm column) with increasing concentrations of EtOAc in ether (9:1 −1:9) and yielded seven fractions (c1−c7). Nematotoxin bioassays (“J2 motility assay”, as described below; concentration = 90 μg/mL) indicated maximum activity resided in fraction c1 (2.56 g), which was then subjected to silica gel chromatography (2.5 × 40 cm column) using ether/EtOAc (100:0−50:50), followed by purification through recycling preparative HPLC (Japan Analytical Industry Co., Tokyo, Japan) using MeOH to afford compound 1 (89 mg), identified as gymnoascole acetate as described under Results and Discussion. Motility and Hatching Bioassays. Nematode Eggs and J2 Preparation. A population of M. incognita originally isolated in Beijing was multiplied in greenhouse pots on tomato plants (Solanum lycopersicum cv. L-402, nematode susceptible). After 45 days, egg masses were handpicked from root galls, surface-sterilized in 0.5% sodium hypochlorite for 3 min, and washed with sterile water three times. Second-stage juveniles (J2s) were hatched from the egg masses in sterile water in 9 cm diameter Petri dishes, collected daily after passage through 25 μm Spectra/Mesh nylon filters (Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA), and stored at 4 °C in sterile water for no more than 2 days. Eggs were extracted with a NaOCl technique21 modified via the use of 0.5% NaOCl and were then poured through a 75 μm pore sieve and collected on a 5 μm pore

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RESULTS AND DISCUSSION Compound 1 was obtained as a white amorphous powder: [α]17D −54 (0.10, CHCl3); UV λMeOH max (log ε) 229 (3.57), 281 (2.12) nm. Its molecular formula, C30H41NO2, was deduced by HRESIMS (m/z 448.3209 [M + H]+, calculated for C30H42NO2, 448.3210) and 13C NMR spectroscopic data, 3128

DOI: 10.1021/acs.jafc.6b04353 J. Agric. Food Chem. 2017, 65, 3127−3132

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Journal of Agricultural and Food Chemistry

carbon resonances comprising 5 methyl (δC 14.9, 17.6, 18.2, 19.5, 26.0), 7 sp3 methylene (δC 22.5, 23.7, 25.0, 26.3, 28.3, 33.3, 38.9), 3 sp3 methine (δC 41.5, 50.2, 77.3), and 5 sp2 methine (δC 112.8, 118.7, 119.7, 120.8, 125.5) carbons, 3 quaternary sp3 carbons (δC 40.3, 41.4, 54.2), and 5 quaternary sp2 carbons (δC 118.0, 126.2, 132.1, 142.1, 152.0). In addition, resonances [δH 2.03 (3H, s); δC 21.1, 172.7] due to an acetoxy group were also observed in the NMR spectra of compound 1. These NMR spectroscopic data were characteristic for an indoloditerpene skeleton with an acetoxy substituent.26−29 The 1 H and 13C NMR spectroscopic data of compound 1 were comparable to those of emindole SB,26 except for the presence of resonances of an acetoxy group (δH 2.03, δC 172.7, 21.1) in compound 1. The deshielded H-16 resonance (δH 4.80, m) indicated that the acetoxy group is located at C-16, which was confirmed by the HMBC correlation between H-16 and the acetoxy carbonyl carbon. The planar structure of compound 1 was further confirmed by the HMBC correlations (Table 1 and Figure 2). The NOESY data revealed that the relative

indicating 11 indices of hydrogen deficiency. The IR spectrum [(KBr) νmax 3413, 2932, 1737, 1636, 1456, 1384, 1244, 1027, 947 cm−1] showed absorption bands for amino (3413 cm−1) and carbonyl (1737 cm−1) functionalities. The 1H NMR spectrum (Table 1) of compound 1 displayed characteristic Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data of Compound 1 (δ in ppm, J in Hz, in Methanol-d4) emindole SB25

compound 1 position

δC, type

1 2 3 4

152.0, C 118.0, C 118.7, CH

5

119.7, CH

6

120.8, CH

7

112.8, CH

8 9 10

142.1, C 126.2, C 28.3, CH2

11 12 13

50.2, CH 26.3, CH2 23.7, CH2

14

δH

7.28 (1H, dd, J = 6.0, 1.5) 6.90 (1H, dt, J = 6.0, 1.0) 6.95 (1H, dt, J = 6.0, 1.5) 7.26 (1H, dd, J = 6.0, 1.0)

HMBC (H→C)

C-3, -6, -8

150.8 118.3 118.4

C-7, -9

119.6

C-4, -8

120.4

C-5, -9

111.4

C-2, -3, -20, -11, -12

41.5, CH

2.30 (1H, dd, J = 11.0, 9.0), 2.61 (1H, dd, J = 11.0, 5.0) 2.76 (1H, m) 1.78 (2H,m) 1.49 (1H, m), 1.65 (1H, m) 1.88 (1H, d, J = 2.0)

15 16

41.4, C 77.3, CH

4.86 (1H, m)

C-15, -17, -23, -24, -30

17

25.0, CH2

18 19 20 21

33.3, 40.3, 54.2, 14.9,

22

19.5, CH3

1.15 (3H, s)

23

18.2, CH3

0.93 (3H, s)

24

38.9, CH2

25

22.5, CH2

26

125.5, CH

1.19 (1H, m), 1.29 (1H, m) 1.85 (1H, m), 1.98 (1H, m) 5.06 (1H, t, J = 6.0)

27 28 29 30 31

132.1, C 26.0, CH3 17.6, CH3 172.7, C 21.1, CH3

CH2 C C CH3

1.80 (1H, m), 1.89 (1H, m) 1.89 (2H, m)

1.03 (3H, s)

δC

C-10, -20 C-11, -12, -14 C-13, -15, -23, -24

140.0 125.2 33.7

49.8 27.5 22.8

Figure 2. Selected HMBC (arrows point from protons to carbons) and NOE correlations of compound 1 (gymnoascole acetate).

configurations at C-11, -14, -15, -16, -19, and -20 of compound 1 are the same as those of emindole SB. In the NOESY spectrum, the NOE correlations of H-11/H3-22/H-13β, H13β/ H3-23, and H3-22/H3-23 indicated that these hydrogens are cofacial and β-oriented, whereas the correlations of H-13α/ H-14 H-14/H3-21, H-14/H-16, and H-16/H2-24 suggested that these protons are α-cofacially oriented (Figure 2). Although quantities were insufficient for specifically determining the absolute configuration, the structure of compound 1 relative to the emindole SB configuration was established as (3S,4S,4aR,6aS,12bS,12cS)-4,12b,12c-trimethyl-4-(4-methylpent-3-en-1-yl)1,2,3,4,4a,5,6,6a,7,12,12b,12c-dodecahydrobenzo[6,7]indeno[1,2-b]indol-3-yl acetate (Figure 1), and the compound was named gymnoascole acetate. Motility bioassay results showed that gymnoascole acetate caused the greatest inhibited motility of M. incognita J2s at all tested concentrations (Table 2). After 24 h of exposure, 95.4% of J2s were immotile at 180 μg/mL (EC50 = 47.5 ± 1.5 μg/ mL); almost all nematodes (97.8%) were immotile after 36 h of treatment (EC50 = 40.6 ± 1.5 μg/mL). At 360 μg/mL, all J2s were immotile by 24 h. Toxicity was proportional to gymnoascole acetate concentration as well as length of exposure. Additionally, the differences in inhibition of motility were significant among different concentration treatments. Gymnoascole acetate treatments also strongly decreased egg hatch of M. incognita (Table 2). Inhibition of egg hatch by gymnoascole acetate concentrations between 6.7 and 267 μg/ mL ranged between 14.6 and 97.2%, with significant differences between all concentrations and the water control. To determine the biological effects of gymnoascole acetate soil application on gall indices and nematode numbers, tomato plants were grown in the presence or absence of nematodes and the compound. The nematode-inoculated control treatment

39.9 39.3 77.3 27.5

C-16, -20, -22

C-2, -11, -19, -20 C-14, -18, -19, -20 C-14, -15, -16, -24 C-14, -15, -16, -23, -25, -26 C-15, -24, -26, -27 C-24, -25, -28, -29

1.67 (3H, s) 1.60 (3H,s)

C-26, -27, -29 C-26, -27, -28

2.03 (3H, s)

C-30

25.2 41.2 53.1 14.6 19.2 16.4 37.5 21.4 124.7 132.2 24.0 17.4

resonances for a 1,2-disubstituted benzene moiety [δH 6.90 (1H, dt, J = 6.0, 1.0 Hz), 6.95 (1H, dt, J = 6.0, 1.5 Hz), 7.26 (1H, dd, J = 6.0, 1.0 Hz), 7.28 (1H, dd, J = 6.0, 1.5 Hz)], an olefinic proton [δH 5.06 (1H, t, J = 6.0 Hz)], and five tertiary methyls (δH 0.93, 1.03, 1.15, 1.60, and 1.67, each 3H, s), as well as an oxygenated methine [δH 4.80 (1H, m)] group. The 13C NMR spectrum (Table 1) along with HSQC data revealed 28 3129

DOI: 10.1021/acs.jafc.6b04353 J. Agric. Food Chem. 2017, 65, 3127−3132

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Journal of Agricultural and Food Chemistry

Table 2. Effect of Gymnoascole Acetate at Different Concentrations on Motility of Second-Stage Juveniles (J2) and Egg Hatch of Meloidogyne incognita in Laboratory Assaysa J2 assays

egg assays

inhibition of motility of J2s (%) concentration (μg/mL) 6.7 9.0 13.3 18.0 33.3 45.0 66.7 90.0 133 180 267 360 0 EC50

12 h

24 h

36 h

4.5 f

6.8 f

8.9 f

21.2 e

32.4 e

38.7 e

40.5 d

48.5 d

57.6 d

51.6 c

65.4 c

65.8 c

88.6 b

95.4 b

97.8 b

98.2 a 0g

100 a 0g

100 a 0g

66.9 ± 1.5

47.5 ± 1.5

40.6 ± 1.5

no. of hatched J2s

hatch suppression (%)

7 days

7 days

91.4 b

14.6

82.0 c

23.4

48.2 d

55.0

26.2 d

75.5

9.0 e

91.6

3.0 f

97.2

107 a

a

Values are means of 6 replicates for J2 assays and 8 replicates for egg hatch assays. Values followed by the same letter in a column do not differ significantly at p = 0.05 according to Duncan’s new multiple-range test.

Table 3. Effect of Gymnoascole Acetate at Different Concentrations on the Numbers of Galls and Second-Stage Juveniles (J2) in Roots and Soil of Tomato Infected with Meloidogyne incognita in Pot Experimentsa galls

J2s in soil

J2s in roots

treatment (μg/mL)

galls/root system

% decrease

J2s/200 g soil

% decrease

J2s/g root

% decrease

2 20 200 0

120 b 85 c 50 d 180 a

33.3 52.8 72.2

287 b 150 c 63 d 426 a

32.6 64.8 85.2

17 b 9c 6d 38 a

55.3 76.3 84.2

a

Values are means of 10 replicates. Values followed by the same letter in a column do not differ significantly at p = 0.05 according to Duncan’s new multiple-range test.

toxicity of indoloditerpenoids upon nematodes has not been examined, use of a Caenorhabditis elegans reversal-of-movement bioassay to quantify BK channel inhibition by penitrem A, emindole SB, and analogues revealed substantial activity by the first but not the second of these compounds.27 Natural products from fungi are a very promising source of new chemicals to manage plant-parasitic nematodes. The EC50 of gymnoascole acetate of 47.5 μg/mL at 24 h is comparable to those of many other nematicidal terpenoids reported from fungi12 and plants.36 The strong and rapid effect of the newly discovered indoloditerpenoid gymnoascole acetate on the rootknot nematode M. incognita indicates that the compound could have some potential use as a bionematicide. However, before definitive conclusions can be reached, field trials would be needed to verify the suitability of the compound in the control of root-knot and other plant-parasitic nematodes.

resulted in the largest numbers of galls and nematodes both in soil and in roots (Table 3). All gymnoascole acetate treatments significantly reduced root galling as well as the J2 population density in the tomato roots and soil compared to the nematode-inoculated control. These results indicate that gymnoascole acetate possesses nematotoxic activity in soil as well as in vitro. Although several indoloditerpenoids have been isolated from fungi, their biological effects have not been extensively studied. A few indoloditerpenoids have insecticidal or insect antifeedant activity; for example, incorporation of penitrem A, an indoloditerpenoid produced by some Penicillium species, at 25 μg/g into the diet of the corn earworm (Heliothis zea) lowered larval weight gains.30 Three penitrem derivatives from Aspergillus sulphureus also reduced H. zea weight gains but were not lethal,31 whereas other indoloditerpenoids (e.g., nominine from Aspergillus nomius) exhibited insecticidal as well as antifeedant activity in H. zea.32 Nodulisporic acid A from a Nodulisporium species exhibited very low LC50 values of 0.3− 0.5 μg/g versus the mosquito Aedes aegypti and the blowfly Lucilia sericata.33,34 Many indoloditerpenoids have attracted interest because of their tremorgenic activity in mammals, which is a result of their strong inhibition of potassium channels.35 Other modes of action demonstrated for this group of compounds include cannabinoid receptor antagonistic activity exhibited by emindole SB.29 Although the direct

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AUTHOR INFORMATION

Corresponding Author

*(D.J.C.) Phone: (301) 504-8634. E-mail: David.Chitwood@ ars.usda.gov. ORCID

David J. Chitwood: 0000-0002-2440-1365 Jun Li: 0000-0001-8243-5267 3130

DOI: 10.1021/acs.jafc.6b04353 J. Agric. Food Chem. 2017, 65, 3127−3132

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This work was partially supported by the Special Fund for Science and Technology Plan Project of Beijing (D151100003915003), the Science and Technology Innovation Fund from Beijing Academy of Agriculture and Forestry Sciences (KJCX20140101), and the National Basic Research Program of China (2013CB127500). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.6b04353 J. Agric. Food Chem. 2017, 65, 3127−3132