Isolation of Secondary Metabolites from the Soil-Derived Fungus

Article pubs.acs.org/JAFC

Isolation of Secondary Metabolites from the Soil-Derived Fungus Clonostachys rosea YRS-06, a Biological Control Agent, and Evaluation of Antibacterial Activity Ming-Ming Zhai,† Feng-Ming Qi,† Jie Li,† Chun-Xiao Jiang,† Yue Hou,† Yan-Ping Shi,‡ Duo-Long Di,‡ Ji-Wen Zhang,§ and Quan-Xiang Wu*,†,‡ †

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China ‡ Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China § Key Laboratory of Botanical Pesticide R&D in Shaanxi Province, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China S Supporting Information *

ABSTRACT: The fungus Clonostachys rosea is widely distributed all over the world. The destructive force of this fungus, as a biological control agent, is very strong to lots of plant pathogenic fungi. As part of the ongoing search for antibiotics from fungi obtained from soil samples, the secondary metabolites of C. rosea YRS-06 were investigated. Through efficient bioassay-guided isolation, three new bisorbicillinoids possessing open-ended cage structures, tetrahydrotrichodimer ether (1) and dihydrotrichodimer ether A and B (2 and 3), and 12 known compounds were obtained. Their structures were determined via extensive NMR, HR-ESI-MS, and CD spectroscopic analyses and X-ray diffraction data. Compounds 1−3 are rare bisorbicillinoids with a γ-pyrone moiety. The biological properties of 1−15 were evaluated against six different Gram-positive and Gram-negative bacteria. Bisorbicillinoids, 2−5, and TMC-151 C and E, 14 and 15, showed potent antibacterial activity. KEYWORDS: Clonostachys rosea YRS-06, antibacterial activity, antifungal activity, tetrahydrotrichodimer ether, dihydrotrichodimer ether, TMC-151

■

INTRODUCTION The fungus Clonostachys rosea (syn. Gliocladium roseum) has a wide geographical distribution. It dwells in the forest, bush, meadow, heathland, desert, and water (from neutral to alkaline). The fungus inhabits subarctic, temperate, subtropical, and tropical regions of the world.1,2 This fungus is also reported as a common saprophyte in soil worldwide.3 The destructive force of this fungus, as a biological control agent, is very strong to lots of plant pathogenic fungi,4 such as Botrytis cinerea sporulation on rose debris,5 major pathogens of cacao,6 strawberry gray mold,7 Fusarium head blight of wheat,8 and mycotoxigenic Fusarium graminearum, which produces the estrogenic mycotoxin zearalenone.9 During the commercial drum priming process, it was used to protect onion and carrot seeds.10 This fungus, as a potential parasite, has significant inhibition activity to nematodes and insects, for instance, sheep nematodes11 and insect pests in greenhouse sweet pepper and tomato.12 The secretion of cell wall degrading enzymes, including chitinases, was related to its antagonistic activity.13 A toxin from the filtrate of C. rosea exhibited potent activity to the nematodes Bursaphelenchus xylophilus, Caenorhabditis elegans, and Panagrellus redivivus.14 However, this fungus has not attracted extensive attention from chemists focusing on the secondary metabolites. Verticillin-type epipolysulfanyldioxopiperazines15 and epidithiodioxopiperazine16 were only recently isolated from G. roseum. © 2016 American Chemical Society

The importance of this fungus in agriculture motivated us to study the C. rosea YRS-06 strain isolated from a soil sample on the bank of the Yellow River in Lanzhou, China. The prominent activities (Table 1) of the crude extracts of this fungus against plant pathogenic fungi, Alternaria alternate (Fr) Keissler, Colletotrichum gloeosporioides, Fusarium graminearum, and Valsa mali Miyabe et Yamada, were investigated. In the ongoing search for antibiotics from fungi derived from soil samples, the antibacterial activities of the extracts toward various Gram-positive (Bacillus megaterium, Bacillus subtilis, Clostridium perf ringens, Micrococcus tetragenus, and a strain of MRSA (methicillin-resistant Staphylococcus aureus)) and Gramnegative (Escherichia coli) human pathogenic bacterial strains were also evaluated. The methanolic crude extracts harvested at 21 days exhibited unexpected results (Table 2). They were subjected to HPLC-UV-ELSD analysis and selected for further purification to yield compounds 1−15 (Figure 1). Several bisorbicillinoids, including three new compounds (1−3), possessing an open-ended cage structure, were presented. Compounds 1−3 are rare bisorbicillinoids with a γ-pyrone moiety. Because of the novel, complex, and compact structures Received: Revised: Accepted: Published: 2298

February 2, 2016 March 7, 2016 March 7, 2016 March 14, 2016 DOI: 10.1021/acs.jafc.6b00556 J. Agric. Food Chem. 2016, 64, 2298−2306

Article

Journal of Agricultural and Food Chemistry Table 1. Antifungal Activity as Inhibition Ratios (%) of Tested Standards and the Extracts inhibition ratios (%) extractsa b

B1 M1c B2b M2c B3b M3c carbendazim

V. mali

C. gloeosporioides

56.49 43.07 77.22 56.10 80.88 75.61 99.80

F. graminearum

15.85 10.85 33.35 18.35 42.10 37.90 98.52

25.63 30.00 44.86 34.62 63.08 52.06 100.00

A. alternate

C. lunata

F. oxysporum

47.00 42.45 68.91 47.73 72.00 70.45 97.33

− − − − − 9.76 100.00

− − 28.41 − 26.94 18.12 98.68

d

a

The concentrations were 100 ppm. bB1 is the extract of the broth for growing 7 d, B2 for 14 d, and B3 for 21 d. cM1 is the extract of the mycelium for growing 7 d, M2 for 14 d, and M3 for 21 d. d“−” means no activity.

Table 2. Antibacterial Activity as MICs (μg/mL) of Tested Standards, the Extracts, and Compounds 1−15 MIC (μg/mL) extracts and compounds

B. megaterium

B. subtilis

C. perf ringens

M. tetragenus

E. coli

MRSA

B1a M1b B2a M2b B3a M3b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 levofloxacin erythromycin ampicillin streptomycin tetracycline

100 100 100 100 50 50 >100 >100 >100 12.5 25 100 100 >100 >100 >100 100 100 100 100 100 6.25 100 50 3.13 3.13

50 100 >100 100 50 50 100 >100 50 100 >100 >100 >100 >100 >100 100 100 50 50 6.25 12.5 25 1.56 12.5 >100 6.25

50 >100 100 100 50 50 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 100 >100 100 25 12.5 12.5 100 >100 100 50

>100 >100 >100 >100 >100 100 >100 >100 >100 >100 >100 >100 100 >100 >100 >100 >100 >100 >100 100 100 100 100 12.5 12.5 6.25

>100 >100 >100 >100 50 50 100 25 50 100 >100 >100 100 >100 >100 >100 100 100 50 3.125 6.25 12.5 50 50 100 6.25

>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100

a B1 is the extract of the broth for growing 7 d, B2 for 14 d, and B3 for 21 d. bM1 is the extract of the mycelium for growing 7 d, M2 for 14 d, and M3 for 21 d.

III-400 (Bruker, Switzerland) and INOVA-600 spectrometers (Varian, USA), using TMS as internal standard. HR-ESI-MS spectra were recorded from an orbitrap Elite spectrometer (Thermo, USA). Singlecrystal data were obtained on a SuperNova, Dual, Eos diffractometer (Agilent, USA). Analytical and semipreparative HPLC were carried out on a 1525 liquid chromatograph (Waters, USA) using the following columns: SunFire (Waters, USA), C18, 5 μm, 150 × 4.6 mm; BDS Hypersil (Thermo, USA), C18, 5 μm, 250 × 4.6 mm; and Luna (Phenomenex, USA), C18, 5 μm, 250 × 10 mm, with 2489 UV/ visible (254 nm) (Waters, USA) and ELSD 6000 (Alltech, USA) peak detections. Silica gel (200−300 mesh) (Qingdao Haiyang Chemical Group Corporation, Qingdao, China) was used for column chromatography (CC). The extraction of the broth was performed on HP20 resin (Mitsubishi, Japan). Precoated silica gel plates (GF254, 10−40 μm, Qingdao, China) was used for thin-layer chromatography (TLC). All solvents were of analytical grade, except for the chromatographic grade used for HPLC.

and the bioactivity, compounds 4−6 made those molecules intriguing targets for chemical synthesis.17−20 To sum up, the isolation and structural elucidation of secondary metabolites from C. rosea YRS-06, a soil-derived fungus, are reported, and the antibacterial activity of 1−15 was evaluated using six different Gram-positive and Gram-negative bacteria. Bisorbicillinoids, 2−5, and TMC-151 C and E, 14 and 15, displayed potent inhibition activity against some of the tested strains.

■

MATERIALS AND METHODS

General. Optical rotations were obtained on an A21202-T digital polarimeter (Rudolph Research Analytical, USA). UV spectra were measured on a UV-2800SPC UV/vis spectrometer (Shanghai, China). IR spectra were recorded on a NEXUS 670 spectrometer using KBr pellets (Nicolet, USA). CD spectra were obtained using a DSM 1000 spectrometer (OLIS, USA). NMR spectra were obtained on AVANCE 2299

DOI: 10.1021/acs.jafc.6b00556 J. Agric. Food Chem. 2016, 64, 2298−2306

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of compounds 1−15. Fungal Material. The fungal strain C. rosea YRS-06 was isolated from a soil sample, which was collected from the bank of the Yellow River in Lanzhou (Loess Plateau, altitude 1520 m, 36°03.821′N, 103°48.404′E), Gansu, China, on Aug 26, 2014. In accordance with the morphological traits and its 18S rRNA sequence (GenBank accession no. KU306742), the fungus was identified. The strain was deposited in a cryogenic refrigerator (−80 °C) in our laboratory. Small-Scale Culture. C. rosea YRS-06 was grown in PS (potato, 20 g; sucrose, 2 g), malt (malt, 2 g; yeast extract, 0.3 g; peptone, 0.5 g; dextrose, 1 g), Czapek (sucrose, 3 g; NaNO3, 0.2 g; K2HPO4, 0.1 g; MgSO4·7H2O, 0.05 g; KCl, 0.05 g; FeSO4, 0.001 g), and YES (sucrose, 15 g; yeast extract, 2 g; MgSO4·7H2O, 0.05 g) liquid media in conical flasks with deionized water (100/250 mL), with shaking (160 rpm) for 7 d in a constant temperature oscillation incubator (26 °C). Extraction and Isolation. The strain (YRS-06, large scale, 60 L) was cultivated in PS liquid medium (potato, 120 g; sucrose, 12 g) in 1 L Erlenmeyer flasks (100) each containing 600 mL of purified water with shaking (160 rpm) for 21 d in two constant temperature oscillation incubators (26 °C). The centrifugal machine (4000 rpm, 20 min) was used to separate the culture broth and the mycelia. The broth (60 L) was extracted with HP20 resin. The HP20 resin extract was orderly washed using water, 50% methanol/water, methanol, and isopropanol. The methanolic extract of the broth (8.3947 g) was yielded. The ground mycelia were extracted with ethyl acetate in an ultrasonicator instrument (3 × 3 L × 30 min, 25 °C). The extract was washed via CC (HP20 resin) with water, 50% methanol/water, and methanol. The methanolic (14.3476 g) extract of the mycelia was obtained. After TLC and HPLC-UV-ELSD analyses (SunFire C18 column (5 μm, 150 × 4.6 mm), 10%−100% methanol/water (0.1% formic acid), 30 min, 1 mL/min), these two methanolic fractions were combined to yield the final extract (22.7423 g). This extract was fractionated via CC (silica gel; chloroform/ methanol at 1:0, 40:1, 20:1, 10:1, 5:1, 3:1, and 1:1) to yield seven fractions (F1−F7). F2 (4.3936 g) was separated via CC (silica gel, chloroform/methanol 20:1) to yield five subfractions (F2-1−F2-5). F2-2 (0.5284 g) was eluted via CC (silica gel, petroleum ether/acetone 6:1 and petroleum ether/ethyl acetate 4:1) to obtain 7 (7.3 mg). F2-3 (3.0000 g) was subjected to CC (silica gel, petroleum ether/ethyl acetate 3:1) to yield two pure compounds, 9 (25.8 mg) and 10 (56.0 mg), and four subfractions (F2-3-1−F2-3-4). F2-3-2 (0.5862 g) was

eluted via CC (silica gel; chloroform/ethyl acetate 6:1, petroleum ether/acetone 4:1, and petroleum ether/ethyl acetate 1:1, separately) to obtain 4 (5.0 mg), 5 (47.1 mg), 6 (20.7 mg), and 11 (4.1 mg). F2-4 (0.3420 g) was divided into five subfractions (F2-4-1−F2-4-5) via CC (silica gel, petroleum ether/ethyl acetate 5:1). F2-4-5 (0.1080 g) was eluted via CC (silica gel, petroleum ether/ethyl acetate 3:2 and chloroform/ethyl acetate 8:3) to obtain 1 (1.9 mg), 2 (4.9 mg), and 3 (5.7 mg). F2-5 (0.3160 g) was separated via CC (silica gel, chloroform/ethyl acetate 4:1) to yield 8 (1.9 mg). F4 (5.4720 g) was divided into three subfractions (F4-1−F4-3) via CC (silica gel, chloroform/acetone 2:1). F4-2 (3.7460 g) underwent further chromatography (silica gel, chloroform/methanol at 7:1 and 5:1) to yield eight fractions (F4-2-1−F4-2-8). F4-2-3 (0.9600 g) was eluted via CC (silica gel, chloroform/methanol 6:1 and ethyl acetate/ methanol 10:1) and HPLC (Luna, C18, 5 μm, 250 × 10 mm; 91% acetonitrile/water (0.1% formic acid) 40 min; 1 mL/min) to obtain 15 (5.3 mg, tR 28.9 min). F4-2-4 (0.1400 g) was eluted via CC (silica gel, ethyl acetate/methanol 6:1) and HPLC (Luna, C18, 5 μm, 250 × 10 mm; 65% acetonitrile/water (0.1% formic acid) 50 min; 1 mL/min) to obtain 14 (6.6 mg, tR 36.1 min). F4-2-6 (0.5000 g) was further eluted via CC (silica gel, ethyl acetate/methanol 7:1) to obtain 13 (6.1 mg). F5 (1.3733 g) was divided into two subfractions (F5-1 and F5-2) via CC (silica gel, chloroform/methanol 5:1). A part of F5-1 (0.9735 g) was used for recrystallization using methanol as the solvent to yield 12 (8.7 mg). Tetrahydrotrichodimer ether, 1: light yellow block crystals; [α]15 D +70.0° (c 0.1, MeOH); CD (Δε) (0.5 mg/mL, MeOH) 275 (−52) and 303 (+50) nm; UV (MeOH) λmax (log ε) 283 (3.93) nm; IR (KBr) νmax 3404, 2918, 2850, 1721, 1656, 1639, 1611, 1511, 1459, 1421, 1265, 1165, 1127, 1017, 966, 739, 704 cm−1; HR-ESI-MS m/z 521.2131 [M + Na]+, calcd for C28H34O8Na, 521.2146; for the 1H and 13 C NMR data, see Tables 3 and 4. Dihydrotrichodimer ether A, 2: light yellow powder; [α]15 D +30.0° (c 0.1, MeOH); CD (Δε) (0.5 mg/mL, MeOH) 273 (−28) and 314 (+14) nm; UV (MeOH) λmax (log ε) 210 (3.66), 284 (3.94), and 358 (3.97) nm; IR (KBr) νmax 3410, 2930, 2855, 1720, 1611, 1546, 1416, 1296, 1262, 1164, 1128, 1035, 963, 870, 736 cm−1; HR-ESI-MS m/z 519.1973 [M + Na]+, calcd for C28H32O8Na, 519.1989; for the 1H and 13 C NMR data, see Tables 3 and 4. 2300


Article

Journal of Agricultural and Food Chemistry Table 3. 1H NMR Data for Compounds 1−3a position 1 8a

1b

9

2.98 (1H, s) 2.46 (1H, dd, J = 14.8, 6.4) 2.60 (1H, dd, J = 14.8, 8.0) 2.26 (2H, m)

10

5.50 (1H, m)

11

5.51 (1H, m)

12 13 14 OH-3 1′ 8′a

1.63 (3H, d, J = 4.0) 1.35 (3H, s) 1.31 (3H, s) 5.57 (1H, s) 3.17 (1H, s) 2.33 (1H, dd, J = 16.8, 3.2) 2.54 (1H, dd, J = 16.8, 13.2) 4.46 (1H, m)

8b

8′b 9′ 10′

12′

5.66 (1H, ddd, J = 15.6, 6.4, 1.2) 5.85 (1H, dq, J = 15.6, 6.8) 1.73 (3H, d, J = 6.4)

13′ 14′ OH-3′

1.24 (3H, s) 1.39 (3H, s) 5.63 (1H, s)

11′

2b

Table 4. 13C NMR Data for Compounds 1−3a 3b

3.11 (1H, s) 6.50 (1H, d, J = 14.8)

3.11 (1H, s) 6.51 (1H, d, J = 15.2)

7.32 (1H, dd, J = 14.8, 10.8) 6.41 (1H, ddd, J = 14.8, 10.8, 1.2) 6.26 (1H, dq, J = 15.2, 7.2) 1.87 (3H, d, J = 6.8) 1.34 (3H, s) 1.31 (3H, s) 5.56 (1H, s) 3.19 (1H, s) 2.28 (1H, dd, J = 16.8, 3.6) 2.52 (1H, dd, J = 16.8, 13.2) 4.32 (1H, ddd, J = 13.2, 6.8, 3.2) 5.57 (1H, ddd, J = 15.2, 6.8, 1.6) 5.75 (1H, ddq, J = 15.2, 1.2, 6.4) 1.66 (3H, dd, J = 6.4, 0.8) 1.24 (3H, s) 1.41 (3H, s) 5.62 (1H, s)

7.31 (1H, dd, J = 14.8, 11.2) 6.42 (1H, ddd, J = 15.2, 10.8, 1.2) 6.25 (1H, dq, J = 14.8, 6.8) 1.87 (3H, d, J = 6.8) 1.33 (3H, s) 1.31 (3H, s) 5.57 (1H, s) 3.18 (1H, s) 2.15 (1H, dd, J = 16.4, 8.4) 2.54 (1H, dd, J = 16.8, 4.4) 4.90 (1H, m)

1b

2b

3b

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′

5.44 (1H, ddd, J = 15.2, 10.8, 1.6) 5.76 (1H, ddq, J = 15.2, 1.2, 6.8) 1.66 (3H, dd, J = 6.4, 0.8) 1.26 (3H, s) 1.39 (3H, s) 5.67 (1H, s)

δ in ppm. Assignments of 1H NMR data are based on the HSQC and HMBC experiments. bRecorded at 400 MHz in acetone-d6. a

59.4 79.6 105.3 59.1 195.5 105.6 192.6 35.2 29.6 130.6 126.7 18.1 22.2 19.6 54.0 79.3 105.1 56.3 173.4 109.8 189.0 41.6 81.2 129.0 131.0 18.0 21.8 19.9

CH C C C C C C CH2 CH2 CH CH CH3 CH3 CH3 CH C C C C C C CH2 CH CH CH CH3 CH3 CH3

59.0 79.5 105.4 60.6 202.0 105.7 175.7 120.7 143.0 132.1 139.9 18.9 22.3 19.7 54.1 79.3 105.0 56.5 173.6 109.6 189.1 41.7 81.4 128.9 131.6 17.9 21.7 19.4

CH C C C C C C CH CH CH CH CH3 CH3 CH3 CH C C C C C C CH2 CH CH CH CH3 CH3 CH3

59.2 79.7 105.5 60.2 202.0 105.7 175.8 120.7 143.0 132.1 139.8 18.9 22.2 19.8 54.7 79.2 105.4 56.0 172.2 108.9 188.7 41.0 80.0 128.4 131.1 18.0 21.8 19.8

CH C C C C C C CH CH CH CH CH3 CH3 CH3 CH C C C C C C CH2 CH CH CH CH3 CH3 CH3

a δ in ppm. Assignments of 13C NMR data are based on the DEPT135, HSQC, and HMBC experiments. bRecorded at 100 MHz in acetone-d6.

Dihydrotrichodimer ether B, 3: light yellow powder; [α]15 D +100.0° (c 0.1, MeOH); CD (Δε) (0.5 mg/mL, MeOH) 273 (−24) and 312 (+21) nm; UV (MeOH) λmax (log ε) 214 (3.54), 278 (3.78), and 357 (3.63) nm; IR (KBr) νmax 3396, 2923, 2854, 1720, 1610, 1459, 1416, 1379, 1263, 1126, 1016, 871, 735 cm−1; HR-ESI-MS m/z 519.1970 [M + Na]+, calcd for C28H32O8Na, 519.1989; for the 1H and 13C NMR data, see Tables 3 and 4. Crystal Data for 1. Light yellow block crystals of 1 were obtained in methanol. Crystal data (CCDC 1449641) were collected with Cu Kα radiation. The structural analysis of 1 was refined with the ShelXL (Sheldrick, 2015) program. The data were collected over a hemisphere of reciprocal space combining with three sets of exposures. At T = 293.89 (10) K the crystal (0.21 × 0.17 × 0.15 mm) was observed to belong to the orthorhombic space group P21212, with a = 23.0058 (10) Å, b = 15.2127 (5) Å, c = 7.8037 (3) Å, V = 2731.12 (18) Å3, Z = 4, Dcalc = 1.212 g/cm3, λ = 1.54184 Å, μ(Cu Kα) = 0.728 mm−1, and F(000) = 1064. The total number of independent refections measured was 6589, of which 4261 were observed, collected in the range of 3.483° ≤ θ ≤ 70.016°. The structure was determined and refined using full-matrix least-squares on F2 values for 3698 I > 2σ(I). All nonhydrogen atoms were anisotropically refined using the least-squares method, and all hydrogen atoms were fixed at calculated positions. The final indices were R = 0.0443, Rw = 0.1147, goodness of fit = 1.008. Antifungal Bioassay. The antifungal activity of the crude extracts in vitro was tested using the poison food technique. Six fungi species, viz., A. alternate (Fr) Keissler, C. gloeosporioides, Curvularia lunata, F. graminearum, Fusarium oxysporum f.sp. vasinfectum (Atk.) Snyder & Hansen, and V. mali Miyabe et Yamada, provided by the Key Laboratory of Botanical Pesticide R&D in Shaanxi Province, Northwest A&F University, were used in the activity test. Carbendazim was employed as a casting positive control, and the extracts dissolved in acetone were screened for antifungal activity. The

test fungi were maintained on PDA medium slants (25 °C), and were cultured in Petri dishes. The Erlenmeyer flasks containing media and Petri dishes were autoclaved for 30 min. The final concentration of the extracts was 100 ppm. Acetone alone served as the control. The media were then poured into a set of two Petri dishes (two replicates) under aseptic conditions in a laminar flow chamber with filter. After partial solidification of the media in the plates, a disk (5 mm diameter) of the fungus was cut from 1-week-old cultured Petri dishes. The treated and control dishes were kept in an incubator (26 ± 2 °C) until the fungal growth completion (2 ± 3 days). The diameter (mm) of the mycelial growth in both treated (T) and control (C) Petri dishes were diametrically measured in three different directions, and the percentage of inhibition (I) was calculated using the following formula: I (%) = [(C − T)/C] × 100. Antibacterial Bioassay. The crude extracts and all of the isolated compounds were evaluated against the bacterial strains, B. megaterium, B. subtilis, C. perf ringens, E. coli, M. tetragenus, and a strain of MRSA, provided by the Key Laboratory of Botanical Pesticide R&D in Shaanxi Province, Northwest A&F University. The positive controls were the antibiotics, ampicillin, erythromycin, levofloxacin, streptomycin, and tetracycline (≥95%) (Sigma, Shanghai, China). DMSO, diluting the extracts and compounds, served as the control. The bacteria were incubated in Mueller−Hinton broth at 30 °C (190 rpm) for 12 h. The final bacterial concentration in the tubes was adjusted to 1 × 105 to 1 × 106 cfu/ML. The minimal inhibitory concentrations (MIC) were determined after incubation at 30 °C for 24 h. The aforementioned test was performed according to the standards of National Committee for Clinical Laboratory. 2301


Article


Figure 2. HPLC-UV-ELSD traces of B1−B3 and M1−M3.

■

250 × 4.6 mm), 20%−100% acetonitrile/water (0.5% formic acid) 30 min and 100% acetonitrile 10 min, 1 mL/min) (Figure 2) was also used for analysis of secondary metabolites. B3 and M3 have more varied chemical contents compared to B1, B2, M1, and M2. After evaluation of the aforementioned data, the methanolic crude extracts harvested at 21 days (B3 and M3) were selected for further purification and yielded 3 new, 1−3, and 12 known, 4−15, compounds. Compound 1, obtained as light yellow block crystals (MeOH), was determined to have the molecular formula C28H34O8 (12 degrees of unsaturation) based on HR-ESI-MS. Its IR spectrum exhibited absorption features that corresponded to alkenyl (1611 and 1459 cm−1), carbonyl (1721 cm−1), and hydroxyl (3404 cm−1) groups. The 1H (Table 3) and 13C NMR (Table 4) spectra in combination with the HSQC spectrum exhibited resonances for six methyls, of which two were secondary [δH 1.63 (3H, d, J = 4.0 Hz, H-12), 1.73 (3H, d, J = 6.4 Hz, H-12′); δC 18.1 (C-12), 18.0 (C-12′)] and four were tertiary [δH 1.35 (3H, s, H-13), 1.24 (3H, s, H-13′), 1.31 (3H, s, H-14), 1.39 (3H, s, H-14′); δC 22.2 (C-13), 21.8 (C-13′), 19.6 (C-14), 19.9 (C-14′)], three were methylenes, seven were methines with four being olefinic [δH 5.50 (1H, m, H-10), 5.66 (1H, ddd, J = 15.6, 6.4, 1.2 Hz, H-10′), 5.51 (1H,

RESULTS AND DISCUSSION The methanolic crude extracts, cultured in PS (broth, 6.4 mg; mycelia, 12.7 mg), malt (broth, 11.9 mg; mycelia, 7.2 mg), Czapek (broth, 5.6 mg; mycelia, 8.2 mg), and YES (broth, 49.5 mg; mycelia, 79.8 mg) (the content ratio of the extract to the pure culture medium is more than 85%), were subjected to TLC analysis, and the PS liquid medium was selected for further fermentation. The strain YRS-06 was grown in a constant temperature oscillation incubator at 26 °C in several conical flasks containing PS liquid medium (600 mL/1000 mL flask) with shaking (160 rpm). The methanolic extracts of the broth, B1 (153 mg) harvested at 7 d, B2 (340 mg) at 14 d, and B3 (525 mg) at 21 d, and of the mycelia, M1 (164 mg) at 7 d, M2 (410 mg) at 14 d, and M3 (850 mg) at 21 d, were subjected to antifungal (Table 1) and antibacterial evaluations (Table 2). The inhibition ratios of B3 and M3 against plant pathogenic fungi, A. alternate, C. gloeosporioides, F. graminearum, and V. mali, were greater than those of B1, B2, M1, and M2. The antibacterial activities of B3 and M3 toward human pathogenic bacterial strains, B. megaterium, B. subtilis, C. perf ringens, and E. coli, were also stronger than those of other extracts. HPLC-UV-ELSD (BDS Hypersil C18 column (5 μm, 2302


Article

Journal of Agricultural and Food Chemistry m, H-11), 5.85 (1H, dq, J = 15.6, 6.8 Hz, H-11′); δC 130.6 (C10), 129.0 (C-10′), 126.7 (C-11), 131.0 (C-11′)], one was oxygenated [δH 4.46 (1H, m, H-9′); δC 81.2 (C-9′)], and two were allylic [δH 2.98 (1H, s, H-1), 3.17 (1H, s, H-1′); δC 59.4 (C-1), 54.0 (C-1′)], and there were 12 quaternary carbons that corresponded to two β-oxygenated-α,β-unsaturated ketones [δC 195.5 (C-5), 105.6 (C-6), 192.6 (C-7), 173.4 (C-5′), 109.8 (C6′), 189.0 (C-7′)], two dioxygenated (sp3-hybridized) [δC 105.3 (C-3), 105.1 (C-3′)], two oxygenated (sp3-hybridized) [δC 79.6 (C-2), 79.3 (C-2′)], and two allylic (sp3-hybridized) [δC 59.1 (C-4), 56.3 (C-4′)].

combination with the large proton coupling value, J8′b,9′ = 13.2 Hz, revealed that H-8′b and H-9′ were at axial positions and H8′a and H-9′ were oriented on the same side of the γ-pyrone ring. However, it was not possible to elucidate the configuration of the double bond (C-10C-11) by the 1H NMR coupling constant. In addition, the relationship between H-9′ of the γpyrone ring and H-1′ and H-14′ was not confirmed by the NOE spectrum. The absolute stereochemistry of 1 was determined according to its CD spectrum and X-ray analysis. Because the bands at 275 (−52) and 303 (+50) in the CD spectrum (Figure 4) were

Figure 3. Key 1H−1H COSY, HMBC, and NOE correlations of compound 1.

The structure of 1 was assigned primarily using the results of the HMBC and the 1H−1H COSY experiments (Figure 3). The 1 H−1H COSY spectrum indicated the presence of fragments of H2-8/H2-9/H-10/H-11/H3-12 (part A) and H2-8′/H-9′/H10′/H-11′/H3-12′ (part B). The correlations from H-1 to C-2, C-3, C-5, C-6, and C-7; from H-13 to C-1, C-2, and C-3; from H-14 to C-3, C-4, and C-5; from H-1′ to C-2′, C-3′, C-5′, C-6′, and C-7′; from H-13′ to C-1′, C-2′, and C-3′; from H-14′ to C3′, C-4′, and C-5′; from H-8′a to C-6′, C-7′, and C-10′; and from H-8′b to C-7′, C-9′, and C-10′, were shown in the HMBC spectrum. These findings constructed a skeletal structure composed of two hydrogenated sorbicillin moieties (parts A and B).21 Parts A and B were combined by direct connections between C-1/C-4′ and C-1′/C-4, which were confirmed via the HMBC correlations from H-1 to C-3′, C-4′, C-5′, and C-14′; from H-14′ to C-1; from H-1′ to C-3, C-4, C-5, and C-14; and from H-14 to C-1′. Their downfield shifts indicated that C-2 and C-2′ were linked to oxygen atoms, and C-3 and C-3′ were connected with two oxygen atoms. Furthermore, the unsaturation degree requirement also supported C-2 being linked to C-3′ and C-2′ to C-3 via two O-bridges to form a dioxane. All of those correlations proved that 1 was a bisorbicillinoid with an open-ended cage structure.22 The unsaturation degree and the molecular formula indicated that one more ring bears a probable γ-pyrone (C-5′−O−C-9′) ring to complete the gross structure of 1. By analysis of 1H NMR coupling constants and the NOE data (Figure 3), the relative configuration of 1 was assigned. Key NOE interactions H-1/H-13/H-14′ and H-1′/H-13′/H-14 indicated that H-1, H-13, and H-14′; H-1′, H-13′, and H-14 were oriented on the same side of each tetrahydrofuran ring (C-1−C-2−O−C-3′−C-4′ and C-1′−C-2′−O−C-3−C-4). The NOE correlations of H-1/H2-8 revealed that C-1 and C-8 were on the same side of the double bond (C-6C-7). The transrelationship between H-10′ and H-11′ was suggested by the J10′,11′ value (15.6 Hz). The NOE correlations of H-8′a/H-9′, in

Figure 4. CD spectra of compounds 1−3.

similar to those of trichodimerol (6) (266 (−14), 317 (+18), and 381 (−15)), dihydrotrichodimerol (5) (271 (−5) and 306 (+6)), and tetrahydrotrichodimerol (4) (279 (−13) and 311 (+55)) possessing a cage structure with the 1S,2S,3R,4R,1′S,2′S,3′R,4′R configuration,22 it is reasonable to consider that cage structure in 1 also has this stereochemistry. These data did not allow the determination of the configuration of H-9′ (R) in the γ-pyrone ring. The structure, including the stereochemistry, was finally confirmed by X-ray analysis (Cu Kα) (Figure 5). It was named tetrahydrotrichodimer ether. Compound 2 was a light yellow powder. The molecular formula was determined to be C28H32O8 (13 degrees of unsaturation) according to the adduct ion peak in the HR-ESIMS spectrum. The alkenyl (1611 and 1416 cm−1), carbonyl (1720 cm−1), and hydroxyl (3410 cm−1) absorption bands were shown in its IR spectrum. Twenty-eight carbon units, including 12 quaternary carbons (six sp3- and six sp2-hybridized), nine methines (three sp3- and six sp2-hybridized), a methylene, and six methyls, were identified via 13C NMR and DEPT spectra (Table 4) in acetone-d6. Its 1H NMR spectrum (Table 3) contained several coupled resonances from δH 5.5 to 7.5 ppm due to the presence of three disubstituted double bonds; δH 1.34 (3H, s, H-13), 1.24 (3H, s, H-13′), 1.31 (3H, s, H-14), and 1.41 (3H, s, H-14′) ppm assigned to four methyl singlets; δH 1.87 (3H, d, J = 6.8 Hz, H-12) and 1.66 (3H, dd, J = 6.4, 0.8 Hz, H-12′) ppm ascribed to a methyl doublet and a methyl quartet, separately; and δH 4.32 (3H, ddd, J = 13.2, 6.8, 3.2 Hz, H-9′) ppm attributed to an oxygenated methine multiplet. Close scrutiny of the aforementioned coupled 1H and 13C NMR data of 2, quite similar to that of 1, revealed that 2 could 2303


Article


the proton at δH 4.90 (1H, m, H-9′), a significant NOE enhancement was obtained at δH 2.54 (H-8′b) in 3, while a NOE correlation between H-9′ and H-8′a was discovered in 2. The configuration of γ-pyrone ring in 3, in contrast to that of 2, could be unambiguously determined via the NOE correlations. Similar bands at 273 (−24) and 312 (+21) in the CD spectrum were also shown (Figure 4). Therefore, compound 3 possesses a cage structure with the 1S,2S,3R,4R,1′S,2′S,3′R,4′R,9′S configuration. It was named dihydrotrichodimer ether B, an epimer (C-9′) of 2. Via comparing the spectroscopic data with the reported data, the isolated known compounds were identified as follows: tetrahydrotrichodimerol,22 4, dihydrotrichodimerol,22 5, trichodimerol,22 6, sorbicillin,23 7, alternariol,24 8, alternariol 5-Omethyl ether,25 9, ergosterol,26 10, (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol,27 11, D-mannitol,28 12, 2-acetamine-2deoxy-α-D-glucose,29 13, TMC-151 C,30 14, and TMC-151 E,30 15. The antibacterial activity (Table 2) of the crude extracts, B3 and M3, against B. megaterium, B. subtilis, C. perf ringen, and E. coli, containing 1−15 supported our hypothesis that they might have comparable activity to the positive controls levofloxacin, erythromycin, streptomycin, tetracycline, or ampicillin. Compound 2 (MIC 25 μg/mL) exhibited stronger antibacterial activity against E. coli than erythromycin, streptomycin, and ampicillin. Compound 3 (MIC 50 μg/mL) exhibited more prominent activity than streptomycin against B. subtilis. Compound 3 (MIC 50 μg/mL) also displayed similar activity against E. coli compared to erythromycin and ampicillin, and greater than streptomycin. The known compound 4 (MIC 12.5 μg/mL) exhibited stronger inhibitory activity against B. megaterium than erythromycin and ampicillin. Compound 5 (MIC 25 μg/mL) also showed significant antibacterial activity against B. megaterium. Compound 12 (MIC 50 μg/mL) displayed stronger activity than streptomycin against B. subtilis. Compound 13 exhibited the same activities as those of 3. TMC-151 C, 14, exhibited stronger antibacterial activities against B. subtilis than levofloxacin, streptomycin, and ampicillin; against C. perf ringens than erythromycin, streptomycin, ampicillin, and tetracycline; and against E. coli than all of the positive controls. TMC-151 E, 15, displayed similar potency to 14. There was not any significant growth inhibition of the crude extracts and the tested compounds to MRSA. In conclusion, this is the first investigation of the secondary metabolites of C. rosea YRS-06, a soil-derived fungus from the bank of the Yellow River in the Lanzhou region in northwest China. Among the isolated compounds 1−15, 3 new compounds, 1−3, were obtained, and their rigid chemical structures were elucidated using NMR, HR-ESI-MS, CD, and X-ray data in the present study. Compounds 1−3 are rare bisorbicillinoids with a γ-pyrone moiety and an open-ended cage structure. The combination of the novel, complex, and compact structures and their bioactivity of compounds 4−6 makes the molecules intriguing targets for chemical synthesis. In biological screening, 2−5 and 13−15 exhibited significant inhibition against several strains of bacteria and could be candidates for antibacterial lead compounds. Compounds 4 and 5 appeared to contribute to the antibacterial property of the extract against B. megaterium, 3 and 12−15 against B. subtilis, 14 and 15 against C. perf ringens, and 2, 3, and 13−15 against E. coli. Because of the potential importance in agriculture and general value of compounds with significant antibacterial

Figure 5. X-ray crystallographic structure of compound 1.

also be a bisorbicillinoid possessing an open-ended cage structure,22 except that two methylenes (CH2-8 and CH2-9) in 1 were replaced with the olefinic methines (δC 120.7 (C-8), 143.0 (C-9) and δH 6.50 (1H, d, J = 14.8 Hz, H-8), 7.32 (1H, dd, J = 14.8, 10.8 Hz, H-9)) in 2. This identification was confirmed via the strong HMBC correlations from H-8 to C-7 and C-9 and from H-9 to C-7, and the 1H−1H COSY correlations H-8 with H-9 and H-9 with H-10. The key NOE interactions H-1/H-13/H-14′, H-1′/H-13′/ H-14, and H-1/H-8 of 2 were the same as those of 1. The transconfigurations of double bonds C-8C-9−C-10C-11 and C-10′C-11′ were elucidated by the J8,9 (14.8 Hz), J9,10 (10.8 Hz), J10,11 (14.8 Hz), and J10′,11′ (15.2 Hz) values. The NOE correlations of H-8′a/H-9′ and the large proton coupling value (J8′b,9′ = 13.2 Hz) were also the same as those of 1, revealing that the two compounds have the same γ-pyrone ring configuration. The bands at 273 (−28) and 314 (+14) in the CD spectrum of 2 (Figure 4) were similar to those of 1. Based on those data, the absolute stereochemistry of 2 was determined and it was named dihydrotrichodimer ether A. Compound 3 was also isolated as a light yellow powder. Analysis of its HR-ESI-MS spectrum resulted in the molecular formula C28H32O8 (13 degrees of unsaturation), which was the same as that of 2. The IR spectrum exhibited absorption features corresponding to alkenyl (1610, 1416 cm−1), carbonyl (1720 cm−1), and hydroxyl (3396 cm−1) groups. Six methyls, a methylene, nine methines, and 12 quaternary carbons were identified by 13C NMR and DEPT spectra (Table 4). Three disubstituted double bonds, four methyl singlets, a methyl doublet and a methyl quartet, and an oxygenated methine multiplet resonance were shown in its 1H NMR spectrum (Table 3). The NMR spectra of 3 were similar to those of 2. Based on these observations, 3 was assumed to possess the same planar structure. On comparison of the 1H NMR data of 3 with that of 2, it was found that the coupling values J8′a,9′ = 3.6 Hz and J8′b,9′ = 13.2 Hz (the large proton coupling value) in 2 were converted to J8′a,9′ = 8.4 Hz and J8′b,9′ = 4.4 Hz. By irradiating 2304


Article


(10) Bennett, A. J.; Mead, A.; Whipps, J. M. Performance of carrot and onion seed primed with beneficial microorganisms in glasshouse and field trials. Biol. Control 2009, 51, 417−426. (11) Baloyi, M. A.; Laing, M. D.; Yobo, K. S. Use of mixed cultures of biocontrol agents to control sheep nematodes. Vet. Parasitol. 2012, 184, 367−370. (12) Kapongo, J. P.; Shipp, L.; Kevan, P.; Sutton, j. C. Co-vectoring of Beauveria bassiana and Clonostachys rosea by bumble bees (Bombus impatiens) for control of insect pests and suppression of grey mould in greenhouse tomato and sweet pepper. Biol. Control 2008, 46, 508− 514. (13) Mamarabadi, M.; Jensen, D. F.; Lübeck, M. An N-acetyl-β-Dglucosaminidase gene, cr-nag1, from the biocontrol agent Clonostachys rosea is up-regulated in antagonistic interactions with Fusarium culmorum. Mycol. Res. 2009, 113, 33−43. (14) Dong, J. Y.; Zhao, Z. X.; Cai, L.; Liu, S. Q.; Zhang, H. R.; Duan, M.; Zhang, K. Q. Nematicidal effect of freshwater fungal cultures against the pine-wood nematode Bursaphelenchus xylophilus. Fungal Divers. 2004, 15, 125−135. (15) Dong, J. Y.; He, H. P.; Shen, Y. M.; Zhang, K. Q. Nematicidal epipolysulfanyldioxopiperazines from Gliocladium roseum. J. Nat. Prod. 2005, 68, 1510−1513. (16) Dong, J. Y.; Zhou, W.; Li, L.; Li, G. H.; Liu, Y. J.; Zhang, K. Q. A new epidithiodioxopiperazine metabolite isolated from Gliocladium roseum YMF1.00133. Chin. Chem. Lett. 2006, 17, 922−924. (17) Barnes-Seeman, D.; Corey, E. J. A two-step Ttotal synthesis of the natural pentacycle trichodimerol, a novel inhibitor of TNF-α production. Org. Lett. 1999, 1, 1503−1504. (18) Nicolaou, K. C.; Simonsen, K. B.; Vassilikogiannakis, G.; Baran, P. S.; Vidali, V. P.; Pitsinos, E. N.; Couladouros, E. A. Biomimetic explorations towards the bisorbicillinoids: total synthesis of bisorbicillinol, bisorbibutenolide, and trichodimerol. Angew. Chem., Int. Ed. 1999, 38, 3555−3559. (19) Nicolaou, K. C.; Vassilikogiannakis, G.; Simonsen, K. B.; Baran, P. S.; Zhong, Y. L.; Vidali, V. P.; Pitsinos, E. N.; Couladouros, E. A. Biomimetic total synthesis of bisorbicillinol, bisorbibutenolide, trichodimerol, and designed analogues of the bisorbicillinoids. J. Am. Chem. Soc. 2000, 122, 3071−3079. (20) Hong, R.; Chen, Y. G.; Deng, L. Catalytic enantioselective total syntheses of bisorbicillinolide, bisorbicillinol, and bisorbibutenolide. Angew. Chem. 2005, 117, 3544−3547. (21) Trifonov, L. S.; Bieri, J. H.; Prewo, R.; Dreiding, A. S.; Hoesch, L.; Rast, D. M. Isolation and structure elucidation of three metabolites from verticillium intertextum: sorbicillin, dihydrosorbicillin and bisvertinoquinol. Tetrahedron 1983, 24, 4243−4256. (22) Liu, W. Z.; Gu, Q. Q.; Zhu, W. M.; Cui, C. B.; Fan, G. T. Dihydrotrichodimerol and tetrahydrotrichodimerol, two new bisorbicillinoids, from a marine-derived Penicillium terrestre. J. Antibiot. 2005, 58, 621−624. (23) Lan, W. J.; Zhao, Y.; Xie, Z. L.; Liang, L. Z.; Shao, W. Y.; Zhu, L. P.; Yang, D. P.; Zhu, X. F.; Li, H. J. Novel sorbicillin analogs from the marine fungus Trichoderma sp. associated with the seastar Acanthaster planci. Nat. Prod. Commun. 2012, 7, 1337−1340. (24) Aly, A. H.; Edrada-Ebel, R.; Indriani, I. D.; Wray, V.; Müller, W. E. G.; Totzke, F.; Zirrgiebel, U.; Schächtele, C.; Kubbutat, M. H. G.; Lin, W. H.; Proksch, P.; Ebel, R. Cytotoxic metabolites from the fungal endophyte Alternaria sp. and their subsequent detection in its host plant Polygonum senegalense. J. Nat. Prod. 2008, 71, 972−980. (25) Tan, N.; Tao, Y. W.; Pan, J. H.; Wang, S. Y.; Xu, F.; She, Z. G.; Lin, Y. C.; Jones, E. B. G. Isolation, structure elucidation, and mutagenicity of four alternariol derivatives produced by the mangrove endophytic fungus No. 2240. Chem. Nat. Compd. 2008, 44, 296−300. (26) Shirane, N.; Takenaka, H.; Ueda, K.; Hashimoto, Y.; Katoh, K.; Ishii, H. Sterol analysis of DMI-resistant and -sensitive strains of Venturia inaequalis. Phytochemistry 1996, 41, 1301−1308. (27) Stonard, R. J.; Petrovich, J. C.; Andersen, R. J. A new C26 sterol peroxide from the opisthobranch mollusk Adalaria sp. and the sea pen Virgularia sp. Steroids 1980, 36, 81−86.

activity, further research should be conducted to identify other hidden properties of soil-derived fungi.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00556.

■

NMR, IR, and HR-ESI-MS spectra of 1−3 and 18S rRNA sequences of C. rosea YRS-06 (PDF) X-ray data of 1 (CIF)

AUTHOR INFORMATION

Corresponding Author

*Fax: +86-931-8915557. E-mail: [email protected]. Funding

This work was financially supported by the National Natural Science Foundation of China (No. 21202075 and 21272103), the 111 Project, the Scientific Research Foundation for Returned Overseas Students (No. 45), the Scientific Research Ability Training of Undergraduate Students Majoring in Chemistry by the Two Patters Based on the Tutorial System and Top Students Project (J1103307). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Toledo, A. V.; Virla, E.; Humber, R. A.; Paradell, S. L.; López Lastra, C. C. First record of Clonostachys rosea (Ascomycota: Hypocreales) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina. J. Invertebr. Pathol. 2006, 92, 7−10. (2) Sutton, J. C.; Li, D. W.; Peng, G.; Yu, H.; Zhang, P.; ValdebenitoSanhueza, R. M. Gliocladium roseum a versatile adversary of Botrytis cinerea in crops. Plant Dis. 1997, 81, 316−328. (3) Schroers, H. J.; Samuels, G. J.; Seifert, K. A.; Gams, W. Classification of the mycoparasite Gliocladium roseum in Clonostachys as C. rosea, its relationship to Bionectria ochroleuca, and notes on other Gliocladium-like fungi. Mycologia 1999, 91, 365−385. (4) Roberti, R.; Veronesi, A.; Cesari, A.; Cascone, A.; Berardino, I. D.; Bertini, L.; Caruso, C. Induction of PR proteins and resistance by the biocontrol agent Clonostachys rosea in wheat plants infected with Fusarium culmorum. Plant Sci. 2008, 175, 339−347. (5) Morandi, M. A. B.; Mattos, L. P. V.; Santos, E. R.; Bonugli, R. C. Influence of application time on the establishment, survival, and ability of Clonostachys rosea to suppress Botrytis cinerea sporulation on rose debris. Crop Prot. 2008, 27, 77−83. (6) Mejía, L. C.; Rojas, E. I.; Maynard, Z.; Van Bael, S.; Arnold, A. E.; Hebbar, P.; Samuels, G. J.; Robbins, N.; Herre, E. A. Endophytic fungi as biocontrol agents of Theobroma cacao pathogens. Biol. Control 2008, 46, 4−14. (7) Cota, L. V.; Maffia, L. A.; Mizubuti, E. S. G.; Macedo, P. E. F.; Antunes, R. F. Biological control of strawberry gray mold by Clonostachys rosea under field conditions. Biol. Control 2008, 46, 515−522. (8) Xue, A. G.; Chen, Y. H.; Voldeng, H. D.; Fedak, G.; Savard, M. E.; Längle, T.; Zhang, J. X.; Harman, G. E. Concentration and cultivar effects on efficacy of CLO-1 biofungicide in controlling Fusarium head blight of wheat. Biol. Control 2014, 73, 2−7. (9) Kosawang, C.; Karlsson, M.; Vélëz, H.; Rasmussen, P. H.; Collinge, D. B.; Jensen, B.; Jensen, D. F. Zearalenone detoxification by zearalenone hydrolase is important for the antagonistic ability of Clonostachys rosea against mycotoxigenic Fusarium graminearum. Fungal Biol. 2014, 118, 364−373. 2305


Article

Journal of Agricultural and Food Chemistry (28) Gaidamauskas, E.; Norkus, E.; Vaičiunienė , J.; Crans, D. C.; ̅ Vuorinen, T.; Jačiauskienė, J.; Baltru̅nas, G. Evidence of two-step deprotonation of D-mannitol in aqueous solution. Carbohydr. Res. 2005, 340, 1553−1556. (29) Coxon, B. Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars. Carbohydr. Res. 2005, 340, 1714−1721. (30) Kohno, J.; Nishio, M.; Sakurai, M.; Kawano, K.; Hiramatsu, H.; Kameda, N.; Kishi, N.; Yamashita, T.; Okuda, T.; Komatsubara, S. Isolation and structure determination of TMC-151s: novel polyketide antibiotics from Gliocladium catenulatum Gilman & Abbott TC 1280. Tetrahedron 1999, 55, 7771−7786.

2306


Isolation of Secondary Metabolites from the Soil-Derived Fungus

Recommend Documents