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Phytotoxic and Antibactrial Metabolites from Fusarium proliferatum ZS07 Isolated from the Gut of Longhorned grasshoppers Shuai Li, Mingwei Shao, Yihui Lu, Lichun Kong, Donghua Jiang, and Ying-lao Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf502484n • Publication Date (Web): 21 Aug 2014 Downloaded from http://pubs.acs.org on August 26, 2014

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Journal of Agricultural and Food Chemistry

Phytotoxic and Antibacterial Metabolites from Fusarium proliferatum ZS07 Isolated from the Gut of Longhorned grasshoppers Shuai Li§,†, Ming-Wei Shao§,†, Yi-Hui Lu†, Li-Chun Kong†, Dong-Hua Jiang†, and Ying-Lao Zhang*,†,‡ †

College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, People’s

Republic of China ‡

State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,

People’s Republic of China

* Corresponding authors. Tel.: +86-579-8228-6419, Fax: +86-579-8228-2269. §

These authors contributed equally to this work.

E-mail addresses: [email protected] (Y. L. Zhang)

1

ACS Paragon Plus Environment


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ABSTRACT In the proceeding of screening new bioactive natural products, ethyl

2

acetate extract of the fermentation broth of Fusarium proliferatum ZS07, a fungus

3

residing in the gut of Longhorned grasshoppers was found possessing selective

4

phytotoxic activity against radicle growth of Amaranthus retroflexus L.

5

Bioactivity-guided fractionation lead to the isolation of six fungal metabolites 1−6,

6

including a new polyketide derivate O-methylated SMA93 (2) and five known

7

compounds SMA93 (1), rhodolamprometrin (3), radicinin (4), dehydroallogibberic

8

acid (5) and 3-methyl-6,8-dihydroxyisocoumarin (6). Their structures were identified

9

on the basis of spectroscopic analysis and by comparison of the corresponding data

10

with those reported in the literature previously. Phytotoxic effects of the four isolated

11

compounds 1-4 on radicle growth of A. retroflexus L. seeds were investigated under

12

laboratory conditions and compounds 2, 4 showed good phytotoxic activity in the

13

concentration of 100 µg/mL, with the inhibition rate of 83.0%, 65.2%, respectively.

14

Furthermore, the antibacterial activity of compounds 1-5 were evaluated against

15

selected bacteria. Compounds 1-3 were found to possess potent antibacterial activity

16

against Bacillus subtilis (ATCC 6633), with the MIC values of 3.13-12.50 µg/mL,

17

while Escherichia coli (ATCC 8739) and Salmonella typhimurium (CMCC(B) 50115)

18

were not susceptible. These results suggest that the new polyketide derivate 2 and

19

known compounds 1, 3, 4 have potential to be used as biocontrol agents in

20

agriculture.

21

KEYWORDS:

22

grasshoppers, phytotoxic activity, polyketide

Antibacterial

activity,

Fusarium

proliferatum,

2


Longhorned

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INTRODUCTION

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Weeds have always been a big problem in agriculture for its affecting crop yield and

25

infesting many types of ecosystems in agriculture.1,2 To control the weeds, synthetic

26

chemicals have been a significant part of management strategies, proved in time to

27

eradicate or control weeds, consequently caused serious problems to public health

28

and brought heavy environmental pollution and weed resistance.3 Considering such

29

restrictions in application of chemicals and the development of new physiological

30

races of pathogens, natural herbicides having low toxicity, high selectivity, and

31

effective activity against weeds was strongly desired.4 Such natural products not only

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may be more environment friendly but also may have novel mode of phytotoxic

33

actions compared to that current suite of herbicides to which weeds are developing

34

resistance.5

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Food safety is another increasingly important public issue in agriculture.

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Consumption of food contaminated with pathogenic bacteria resulted many cases of

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human illness such as vomiting and diarrhoea.6 Moreover, microorganisms are

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associated with food spoilage, causing economic losses every year.7 Technologies

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like activated films, irradiation and synthetic additives were applied to avoid

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microbial growth. However, these procedures caused loss of organoleptic properties

41

of foods and produced adverse health effect.8 Therefore, there is growing interest to

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develop new methods of eliminating food borne pathogens. One such possibility is

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the use of metabolites produced by microbes.9,10

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Insect gut microbes participating in parasitic or commensal relationships with

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their hosts are rich and complex microorganisms communities, which have received

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considerable attention as a resource for novel bioactive metabolites.11,12 However,

47

only a small percentage of such diversity groups have been cultivated and

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chemically studied.3 In the course of our ongoing efforts to screen new bioactive

49

metabolites from the insect gut microbiota, we found that the ethyl acetate extract

50

from the culture filtrates of the fungal strain Fusarium proliferatum ZS07, isolated

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from the gut of Longhorned grasshoppers, exhibited potent phytotoxic activity

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against the radical growth of Amaranthus retroflexus L, and antibacterial activity to

53

four food spoiling microorganisms. Further investigation of the crude extract

54

resulted in the isolation of one new polyketide derivate along with 5 known

55

compounds. Here, we reported the details of the isolation, structure elucidation, and

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biological activities of the metabolites.

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MATERIALS AND METHODS

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Isolation and Identification of Strain ZS07

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The fungal strain was isolated according to the methods detailed previously.13

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Healthy grasshoppers (L. grasshoppers) were collected from the suburb of Jinhua,

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Zhejiang Province, PR China. The samples were transported to the laboratory and

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hungered for 24 h. Samples were sterilized in 75% ethanol for 2 min followed by

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rinsing three times in sterilized water, then the grasshoppers were degutted using

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sterile forceps. The guts were homogenized, and dilution series (10-1, 10-2, 10-3) were

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spread-plated on malt-extract agar (MEA) medium (consisting of 20 g malt extract,

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20 g sucrose, 1 g peptone, 20 g agar in 1 L of distilled water). The plates were

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incubated aerobically in a chamber for 72 h at 28 ± 0.5 °C and colonies were

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transferred and purified on new MEA mediums to obtain pure cultures.

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The fungal strain was differentiated by the morphological characteristics according

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to the manual description of Cappuccino and Sherman. Genomic DNA was extracted

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and ITS regions sequence was amplified using the universal primers ITS1 and ITS4.

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Then the sequence was submitted to GenBank (accession no. KJ490634) and

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analyzed by BLAST search with reference sequences. Phylogenetic analysis was

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done by neighbor-joining in MEGA 5.0 with 1,000 bootstrap replicates. The strain

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was deposited at China Center for Type Culture Collection (CCTCC) as M2013257.

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Fermentation

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The strain was cultured on MEA medium at 28 ± 0.5 °C for 3 days until the colony

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emerged. Then pieces of fresh mycelium were inoculated into 250 mL Erlenmeyer

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flasks each containing 100 mL of ME liquid medium. After 2 days of incubation at

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28 ± 0.5 °C on rotary shakers at 150 rpm. 20 mL suspension of the strain was

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transferred as seed into 1 L Erlenmeyer flasks each containing 500 mL of ME liquid

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medium. The flask cultures were incubated at 28 ± 0.5 °C for 7 days.

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Phytogrowth Inhibitory Bioassay of Ethyl Acetate Extracts

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The phytotoxic effects of the ethyl acetate extracts of ZS07 were evaluated on

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radicle growth of A. retroflexus L. and selected crops (Brassica campestris L.,

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Glycine max, Lycopersicon esculentum Mill., Capsicum annuum) easily to be

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affected by A. retroflexus L. on the base of petri dish bioassay.14 Briefly, seeds were

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first pretreated with sodium hypochlorite (1%) and washed with sterile distilled

5



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water before germination. Then 25 pre-germinated seeds were placed in 9 cm

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diameter petri dishes on filter paper disks imbibed with 5.0 mL sample solution (100

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µg/mL). To avoid toxic effect of solvents, filter papers were placed in a cabinet to

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evaporate the solvent. Subsequently, 5.0 ml of distilled water was added to each petri

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dish. 2,4-dichlorophenoxy acetic acid (2,4-D) was used as the positive control and

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three replicates were prepared for each sample. Dishes were then kept in a growth

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chamber at 25 °C under continuous light. After 2 days, root length were measured

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and compared to the proper untreated control. The inhibition percent15 was calculated

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using the formula below.

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Inhibition (%) = (Lcontrol-Ltreatment)/Lcontrol × 100

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where Lcontrol = radicle length of seedlings in the control.

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Ltreatment = radicle length of seedlings treated.

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Isolation and Characterization of Secondary Metabolites

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A total of 40 L of fermentation broth was filtered and extracted three times with an

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equal volume of EtOAc at room temperature. The solvent was then removed in

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vacuo to give a crude extract (3.0 g). The extract was subjected to a silica-gel

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column eluting with a stepwise gradient of CH2Cl2/MeOH (100:0-100:4, v/v) to

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afford four fractions (Fr-1 to Fr-4). Fr-1 was further chromatographed over silica gel

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(CH2Cl2/MeOH, 100:0−100:2) to give five subfractions (R1-R5), compound 2 (30

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mg) was crystallized from the MeOH solution of subfraction R2, the remaining

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fraction of R4 and R5 were combined and purified by Sephadex LH-20 using a

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CH2Cl2/MeOH mixture (1:1) as the eluent to give the compound 6 (3 mg). Fr-2

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(CH2Cl2/MeOH, 100:1) was repeatedly purified on Sephadex LH-20 (MeOH) and

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detected by TLC to yield compound 4 (6 mg). Fr-3 (CH2Cl2/MeOH, 100:2) was also

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loaded onto a Sephadex LH-20 column (MeOH) to give compounds 3 (35 mg), 5 (5

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mg) and 1 (50 mg).

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Structural identifications of the secondary metabolites were made by the

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spectroscopic analysis. 1H nuclear magnetic resonance (NMR),

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distortionless enhancement by polarization transfer (DEPT) spectra were measured

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with a Bruker AVANCE-400 (Bruker, Switzerland) spectrometer at 400 MHz and

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chemical shifts were reported as parts per million (δ) by referring to the solvent

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signals and tetramethylsilane (TMS) as internal standards. 1H and

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assignments were supported by the 1H-1H COSY, HMQC and HMBC experiments.

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The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired on a

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Mariner Mass 5304 instrument.

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Phytogrowth Inhibitory Bioassay of Metabolites

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The phytotoxic effects of compounds 1-4 were evaluated on radicle growth of A.

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retroflexus L. as described above. Concentrations of compounds 1-4 at 1, 10, and

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100 µg/mL were prepared with acetone. Then, 5.0 mL of varying concentrations

128

solution was applied to each Petri dish. After the solvent was evaporated, 5.0 mL of

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distilled water was added to the Petri dish before the sowing of pre-germinated 25

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seeds of A. retroflexus L. Root length were measured and 2,4-D was used as the

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positive control.

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Detection of Antibacterial Activity.

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13

C NMR and

13

C NMR


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The disc diffusion method was employed for the determination of antibacterial

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activity of the bioactive metabolites. Bacterial strains Escherichia coli (ATCC 8739),

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Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538) and Salmonella

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typhimurium (CMCC(B) 50115) were cultured overnight at 37 °C in Mueller Hinton

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broth (MHB) and then 0.2 mL suspension of the tested microorganisms (1.0×108

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cfu/mL) were spread on the solid media plates. Filter paper disks containing 5 µL of

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each metabolite solution (6 mg/mL) were applied to the surface of agar plates. The

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plates were incubated at 37 °C for 18 h. The diameters of the inhibition zones were

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measured and average diameter values calculated for each compound. All tests were

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performed in triplicate. The minimum inhibitory concentrations (MICs) of purified

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compounds against bacteria were determined using the microbroth dilution method16

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in disposable 96-well microtiter dishes. A stock solution of each sample was

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prepared at a concentration of 200 µg/mL in MHB (supplemented with 1% DMSO

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and 4% Tween 80, v/v), which was further 2-fold diluted and micropippetted

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separately into individual microplate wells (100 µL/well) with a series of

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concentrations range from 100 to 0.78 µg/mL. Then, a standard amount of the tested

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microbes (1.0×106 cfu/mL) were added per well and incubated 24 h at 37 °C for E.

150

coli, S. aureus, B. subtilis, S. typhimurium. The antibacterial activity was evaluated

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by comparing with a control well containing culture broth and microorganisms

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without the compound. The MIC was defined as the minimum concentration of

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compounds at which the bacterial growth was inhibited, as indicated by the absence

154

of turbidity. Each test was performed in duplicate and gentamicin was served as

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reference compound.

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RESULTS AND DISSCUSSION

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Identification of the Fungus

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Morphological characteristics of the fungus were observed in potato dextrose agar

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(PDA) medium. The colonies were slow growing at first, becoming floccose with

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age and purple in color. Reddish-purple appeared in the reverse of colony when

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aerial mycelium reached the edge of medium. Sporulation started early in aerial

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mycelium. Microconidia were produced in false head and variable in shape.

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Conidiophores originated erect from the substrate, sympodially branched bearing

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nearly cylindrical monophialides. The isolated fungus would be located in the

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Liseola group and showed high similarity in morphology with Fusarium.

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Phylogenetic taxonomy with sequence alignment of ITS-rDNA of the fungus was

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done with MEGA 5.0 software. The phylogenetic tree (Fig. 1) indicated that the title

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fungus was closely related to F. proliferatum (FJ648201), with the ITS sequence

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similarity of 99.5%. Combined with the morphological characteristics, the fungus

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was identified as F. proliferatum.

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Phytotoxic Activity of the Ethyl Acetate Extracts

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Petri dish bioassay was used to evaluate the phytotoxic activities of ethyl acetate

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extract from F. proliferatum against radicle growth of A. retroflexus L and selected

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crops. The results (Fig. 2) indicated that the crude extract showed selectivity greater

175

than 2 times for weed over the tested crop. Under the concentration of 100 µg/mL,

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the crude extract produced significance inhibition against the radicle growth of A.

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retroflexus L with the inhibition rate of 67.6%, while it produced weak inhibition

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against the radicle growth of B. campestris L., G. max, L. esculentum Mill and C.

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annuum with inhibition rate of less than 35%. Our research provided evidence that

180

the extract of F. proliferatum showed good selective phytotoxic activities for the A.

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retroflexus L.

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Identification of Active Compounds

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Bioassay-guided fractionation of the constituents in the ethyl acetate extract of F.

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proliferatum ZS07 yielded a novel derivate 2 and five known active metabolites (Fig.

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3).

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Compound 2 was obtained as an orange powder and its molecular formula

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C19H16O7 was deduced from HR-ESI-MS (m/z 379.0789 [M + Na]+, calcd for

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C19H16O7Na 379.0794), which was consistent with the 1H NMR and 13C NMR data.

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The 1H NMR (Table 1) of 2 indicated the presence of 1,3-disubstituted benzene ring

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(δH 6.13, J = 2.3 Hz; δH 6.23, J = 2.3 Hz), one methoxyl (δH 3.87), one methyl (δH

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2.14) and one methylene (δH 4.14). Three proton signals at δH 10.96, 10.26, 9.79

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were assigned to hydroxy groups because no HMQC correlation was observed. The

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1

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position 5 in 2 appeared to be OCH3, which was corresponding to the increase in

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molecular weight of 2 by 14 amu compared to 1. This was further confirmed by the

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HMBC correlation of MeO-5 to C-5 (δC 167.0). Further confirmation was achieved

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by the HMBC correlation of H-4 to C-2 (δC 99.6), C-3 (δC 163.0), C-5, C-6 (δC

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101.9); H-6 to C-2, C-5, C-7 (δC 139.7), C-8 (δC 107.2); H-10 to C-8, C-9 (δC 152.9),

H and 13C NMR data were similar to those of SMA93 (1)17 except the substituent at

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C-11 (δC 199.7); H-14 to C-12 (δC 119.2), C-13 (δC 158.5), C-15 (δC 160.5), C-16 (δC

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110.0); H-16 to C-12, C-14 (δC 100.7), C-17 (δC 139.7), and H-18 to C-16, C-17.

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Thus, the structure of 2 was determined as an O-methylated derivative of 1.

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The other secondary metabolites were identified as rhodolamprometrin (3),18,19 (4),3

dehydroallogibberic

acid

(5)20,21

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radicinin

and

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3-methyl-6,8-dihydroxyisocoumarin (6),22 by spectroscopic data analyses and

205

comparison of their or derivative data in the literature.

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The genus of Fusarium species was well-known to produce a variety of secondary

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metabolites. Previous investigations led to the isolation of several compounds such as

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toxic fumonisins,23,24 mycotoxins,25 antibiotic and insecticidal beauvericin,26 HDAC

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inhibitor apicidin27 and other compounds related to the precursors.19 However, to our

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best knowledge, it was the first report that the new polyketide 2 and the following

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metabolites1, 3, 4, 5 were isolated from the title strain F. proliferatum ZS07, a fungus

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residing in L. grasshoppers gut.

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Phytotoxic Activity of the Bioactive Metabolites.

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Compounds 1-4 were assayed for their ability to inhibit radicle growth of A.

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retroflexus L. using a petri dish bioassay. The result (Fig. 4) showed that compound

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2 was very active to reduce radicle growth of A. retroflexus L. under the

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concentration of 100 µg/mL. With a little morphological alterations of the radicle,

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the relative inhibition rate of compound 2 was 83.0%, which was comparable to that

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of positive 2,4-D with the inhibition rate of 86.4%. The compound 4 showed

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moderate phytotoxic activity with the inhibition rate of 65.2% at the concentration of

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100 µg/mL. However, compounds 2, 4 showed weak inhibitory effect in a low

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concentration. No obvious inhibitory effects were displayed by the compounds 1, 3

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in this bioassay.

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Antibacterial Activity of the Bioactive Metabolites.

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The disc diameters of zone of inhibition (ZOI) and MIC values of compounds 1-5

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against different bacteria were presented in Table 2. The results showed that

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compound 3 had the greatest antibacterial effect against B. subtilis with ZOI of 24.8

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mm and MIC value of 3.13 µg/mL, which were comparable to those of referenced

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gentamicin with the ZOI and MIC value of 25.6 mm, 3.13 µg/mL, respectively.

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Similar with the inhibition zone result of 19.5 mm, compound 1 possessed moderate

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antibacterial effect against B. subtilis with MIC value of 6.25 µg/mL, followed by

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the new derivate 2 with MIC value of 12.50 µg/mL. The replacement of phenolic

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hydroxyl group by one methoxy group in the phenolic nucleus of 1 resulted in the

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weaker antibacterial activity for B. subtilis, which was in agreement with the earlier

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postulate.28 Compounds 4, 5 inhibited the growth of B. subtilis in disc diffusion tests,

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but the MIC values of them were not detected in the concentrations of 100 µg/mL.

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Similarly, compounds 1, 2, 3 possessed weak antibacterial activities against S.

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aureus in disc diffusion tests, but no inhibition was found in MIC test. The

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remaining microorganisms E. coli and S. typhimurium were not susceptible to all

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compounds.

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In summary, we identified one new polyketide, together with five known

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compounds, from a fungus F. proliferatum ZS07. The new polyketone 2 and the

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known compound 4 attenuated the radicle growth of A. retroflexus L. and for the first

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time, we found that compounds 1, 3 possessed potent or moderate antibacterial

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activity against B. subtilis in vitro. These results suggested that the compounds 1-4

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have some potential as agents for weeds and pathogenic bacteria control. Further

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studies will be carried out to better understand the mechanism of action associated

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with phytotoxic and antibacterial effects. In addition, the discovery of our study

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provided additional evidence that uninvestigated habitats, just like the title strain, may

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inspire the discovery of chemical agents with interesting biological activity.

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Funding

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This work was co-financed by the National Natural Science Foundation of China

253

(NSFC) (21002092 and 21272215) and Open Project of State Key Laboratory of

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Pharmaceutical Biotechnology in Nanjing University (KF-GN-201411).

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Notes

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The authors declare no competing financial interest.

257

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Chem. Soc. C, 1971, 2980-2983.

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Figure Captions: Figure. 1. Phylogenetic tree of the fungus ZS07 based on the 5.8S rDNA sequences. Figure. 2. The phytotoxic effects of crude extract of ZS07 on radicle growth of A. retroflexus L. and tested crops (100 µg/mL). Figure. 3. Chemical structures of secondary metabolites 1-6 of F. proliferatum ZS07. Figure. 4. The phytotoxic effects of compounds 1-4 on radicle growth of A. retroflexus L.

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Table 1. 1H NMR and 13C NMR data of compound 2 in DMSO-d6 Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 3-OH 5-OCH3 13-OH 15-OH

δH, mult. (J in Hz)

6.55, d (2.3) 6.63, s 6.62, d (2.3) 4.14, s

6.23, d (2.3) 6.13, d (2.3) 2.14, s 10.96, s 3.87, s 10.26, s 9.79, s

δC 165.7 99.6 163.0 101.1 167.0 101.9 139.7 107.2 152.9 48.4 199.7 119.2 158.5 100.7 160.5 110.0 139.7 20.8 56.4

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Table 2. Zone of inhibition (mm), MIC (µg/mL) of compounds 1-5 against the tested bacteria B. subtilis Compound

S. aureus

ZOIa

MIC

ZOI

MIC

1

19.5 ± 0

6.20

11.7 ± 0.5

＞100

2

13.8 ± 0.6

12.50

8.4 ± 0.4

＞100

3

24.8 ± 0.6

3.13

8.0 ± 0

＞100

4

6.6 ± 0

＞100

NI

＞100

5

9.2 ± 0.6

＞100

NI

＞100

GENb

25.6 ± 0.5

3.13

30.0 ± 0

3.13

a

ZOI: Zone of inhibition, results were presented as mean ± standard deviations for triplicate experiments.

b

GEN: gentamicin 30 µg/disc.c NI: no inhibited.

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Figure. 1.

21



Figure. 2.

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OH

O 1

3

O

O

O

HO

9

R 5

O O

OH

OH O

7 11

17

O

O 1 R= OH 2 R= OCH3 HO 13 OH

OH 15 OH

O

OH 4

3 HO O

OH O

OH

5

O 6

Figure. 3.

23



Figure. 4.

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TOC Graphic

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Phytotoxic and Antibacterial Metabolites from ... - ACS Publications

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