徐利剑,李培琴,赵江林,单体江,阴春晖,王明安,周立刚＊

1黑龙江大学农业资源与环境学院,哈尔滨 150000; 2中国农业大学农学与生物技术学院;3中国农业大学理学院,北京 100193

Introduction

Microorganis ms,in particular fungi,are known as a rich source ofnoveland unique antimicrobialcompounds[1,2].Fungal endophytes are the fungiwhich live inside the healthy plant tissue,have been assumed to be a rich source of natural products such as plant growth regulators,ant imicrobial agents and insecticides[3,4].In the previous study,we isolated and characterized nine distinct fungal endophytes from the rhizomes ofD ioscorea zingiberensis,a traditional Chinese medicinal plant[5].Of them,the n-butanol extract of Fusarium redolensDzf2 showed strongest antimicrobial activity.One antimicrobial compound has been isolated and identified as beauvericin from F.redolens[6].In this study,two antimicrobial compounds were further obtained from the n-butanol extract ofF.redolensDzf2 by bioassay-guided fractionation,their antimicrobial activitieswere also evaluated.

Materials andM ethods

General

ESI-MS was perfor med on a Ther mo Finnigan LCQ mass spectrophotometer.NMR spectra were recorded on a Bruker NMR spectrometer(1H:300 MHz;13C:75 MHz)using T MS as the internal standard.The melting point was determined on an XT4-100 micro-melting point apparatus(Tianjin Tianguang Optical Instruments Company,China) and uncorrected.The microplate spectrophotometer(Powe rWave HT,BioTek Instruments,USA)was employed to measure the light absorption values.Silica gel(200-300 mesh)for column chromatography(CC)and silica gel GF254for TLC were products of Qingdao Haiyang Chemical Company,China.Sephadex LH-20 and silica gel RP-18 were from Pharmacia Biotech,Sweden.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)was from Amresco.All chemicals used in the study were of analytical grade.

M icroorgan is m s

EndophyticF.redolensDzf2 was one of the nine endophytic fungal strains isolated from the healthy rhizomes ofD.zingiberensisand it showed stronger antimicrobial activity than any other fungal endophytes fromD.zingiberensis[5].One plant pathogenic fungus(M agnaporthe oryzae), two gram-positive (Bacillus subtilis ATCC11562 and Staphylococcus haem olyticus ATCC29970)and four gram-negative(Agrobacterium tum efaciensATCC11158,Pseudom onas lachrym ans ATCC11921,Escherichia coliATCC25922 andXanthom onas vesicatoriaATCC11633)bacteria were used as testmicro-organis ms.

Isolation and purification

The 1000-mL volume Erlenmeyer flasks containing 500 mL of liquid PD(potato dextrose)medium were inoculated and each waswith 2-3 agar plugs taken from cultures ofF.redolensDzf2 isolates purified on PDA(potato dextrose agar).All shake flasks were incubated at 120 rpm on a rotary shaker at 25°C for 7 days.After suspension culture,40 L of culture broth was separated from the mycelia by filtration in vacuum.Culture filtrateswere extracted with an equal volume of n-butanol for three times.The remaining mycelia were dried and ground with a mortar and pestle,followed by sonicating and extractingwith 2 L of n-butanol for three times(3 ×2 L).The n-butanol solutions were concentrated in vacuum at 50°C to obtain mycelia and culture filtrate extracts,respectively.The mycelia extracts and the culture filtrate extractswere combined as their TLC results were similar.The extract(47 g)was separated over silica gel(200-300 mesh)column eluted gradiently with ethyl acetate/methanol(10:0,9:1,8:2,7:3,5:5,3:7, 1:9,0:10,v/v)to yield eleven fractions(F-1 to F-8). F-8(12.09 g)from the eluate of methanol showed obvious antimicrobial activity and it was selected for further isolation of ant imicrobial compounds.Two ant imicrobial compounds 1(8 mg)and 2(15 mg)were obtained by repeated column chromatography over silica gel,RP-18 gel and SephadexLH-20 combined with the TLC-bioautographic assay[7].

Structural identification

Fusaric acid(1) was obtained as white needle crystal(MeOH),mp.97-100℃.Its molecular formula was established as C10H13NO2on the basis of the molecular ion peak at m/z 180[M+H]+of ESI-MS.1H NMR (MeOD,300 MHz)δ(ppm),8.66(1H,d,J=2.5 Hz,H-6),8.18(1H,d,J=8.0 Hz,H-3),7.79(1H, dd,J=8.0,2.0 Hz,H-4),2.75(2H,t,J=7.5 Hz,H-7),1.66(2H,m,J=7.5 Hz,H-8),1.38(2H,m,J= 7.5 Hz,H-9),0.94(3H,t,J=7.5 Hz,H-10).The mp.,ESI-MS and NMR data were identical to those reported[8-10].9,10-Dehydrofusaric acid(2)was obtained aswhite needle crystal(MeOH),mp.105-108℃.Its molecular formula was established as C10H11NO2on the basis of the molecular ion peak atm/z178[M+H]+of ESI-MS.1H NMR(MeOD,300 MHz)δ(ppm),8. 51(1H,d,J=1.2 Hz,H-6),8.10(1H,d,J=7.8 Hz, H-3),7.88(1H,dd,J=7.8,1.2 Hz,H-4),5.84 (1H,m,H-9),5.00(2H,q,H-10),2.84(2H,t,J= 7.5,H-7),2.43(2H,q,J=6.6,H-8);13C NMR (MeOD,75 MHz)δ(ppm),167.50(COOH),147.28 (C-2),125.96(C-3),139.82(C-4),143.21(C-5), 149.68(C-6),33.15(C-7),35.74(C-8),138.09(C-9), 116.46(C-10).The mp.,ESI-MS and NMR data were identical to those reported[8-10].

Fig.1 Fusaric acid(1)and 9,10-dehydrofusaric acid (2)from endophytic fungusFusarium redolensDzf2

Ant im icrobial assay

To evaluate the median effective inhibitory concentration(IC50)of the compounds against bacteria,a colorimetric assay by using chromogenic reagent 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT)was employed[11].Briefly,all wells of the sterile 96-wellmicroplate were filled with 90μL test bacterial suspension containing 106 cfu per milliliter.Test samples(10μL)with their different concentrations were added into each well.After the culture plate was incubated for 24 h at 28°C,10μL ofMTT stock solution at concentration of 5 mg/mL was added into each well,then the plate was incubated for another 4 h at 28℃.The reaction was stopped by adding 150μL ofDMSO into each well.After incubation for 30 min with slight shaking on a microplate shaker at 28℃,the plate was centrifuged for 10 min at 3000 g.100μL of the supernatant in each wellwas transferred to a corresponding well of another 96-wellmicroplate to measure their light absorption values at wavelength 510 nm using a microplate spectrophotometer.All testswere perfor med in triplicate.The percentage(%)of bacterial growth inhibition was calculated from mean values using the for mula[(Ac-At)/Ac]×100,where Ac was the average absorbance at wavelength of 510 nm with the negative control,and Atwas the average absorbance at wavelength of 510 nm with sample treatment.The median effective inhibitory concentration(IC50)against bacteria was calculated using the linear relation between the inhibitory probability and concentration logarithm.

The spore germination inhibition assay was selected to test antifungal activity[12].The spores ofM.oryzaewere harvested from cultureson the oatmeal-tomato agar,and they were diluted to 106spores/mL suspension with sterile water.The compounds were diluted to different concentrations with 20%ethanol.The spore ger mination inhibition assay was carried out on the glass slide with a well and each well inoculated 25μL of compound solution and 25μL of spore suspension.The glass slide was cultured at 25℃for 7 h in a 9-cm diameter Petri dish for maintaining humidity.300 spores were counted for germination status,and germination rate was determined on the basis of mean values of three repeats.IC50values could be obtained based on the spore germination rate with different concentrations of each compound,and calculated the same as that for antibacterial activity assay.

Results and D iscussion

Two compoundswith antimicrobial activitieswere isolated from endophyticF.rendolensDzf2 extracts and identified as fusaric acid(1)and 9,10-dehydrofusaric acid(2)on the basis of spectral and physicochemical data(Fig.1).The bio-guided isolation was carried out by using antimicrobial detection on the TLC plate with bio-autographic method[7].TLC-bioautographic detection combined the chemical property such as visualization on TLC plate offers more infor mation for the antimicrobial compound separation.

Fusaric acid and 9,10-dehydrofusaric acid have ever been isolated fromFusarium nygam ai,which could inhibitweed seed germination[9].According to the previous studies on fusaric acid,there was a dispute about whether fusaric acid involved in plant wilt pathogenic process(e.g.tomato)[13,14].It was found that fusaric acid wasmildly toxic to mice[15].

The two compounds had strong ant imicrobial activities against six bacteria and one fungus(Table 1).The antimicrobial activities of 9,10-dehydrofusaric acid were a little stronger than those of fusaric acid except onX. vesicatoria.It implied that the introduction of double bond atC-9 of fusaric acid could increase its antimicrobial activity.The crude extract of the endophyticF. redolenswas screened to have strong antimicrobial activity according to our previous report[5].It suggested that fusaric acid and 9,10-dehydrofusaric acid should be the active compounds in endophyticF.redolens,suggesting the potential application ofF.redolens.

Table 1 Ant im icrobial activity of fusaric acid(1)and 9, 10-dehydrofusaric acid(2)

＊The positive control for the bacteria was streptomycin sulfate,and that forM.oryzaewas carbendazim.

1 KnightV,Sanglier JJ,Ditullio D,et al.Diversifyingmicrobial natural products for drug discovery.ApplM icrobiol B iotechnol,2003,62:446-458.

2 PapagianniM.Fungal morphology and metabolite production in submerged mycelial processes.B iotechnol Adv,2004,22: 189-259.

3 Tan RX,Zou WX.Endophytes:a rich source of functional metabolites.Nat Prod Rep,2001,18:488-459.

4 Xu L,Zhou L,Zhao J,et al.Recent studies on the antimicrobial compounds produced by plant endophytic fungi.Nat Prod Res Dev(天然产物研究与开发),2008,20:731-740.

5 Xu L,Zhou L,Zhao J,et al.Fungal endophytes fromD ioscorea zingiberensisrhizomes and their antibacterial activity. Lett ApplM icrobiol,2008,46:68-72.

6 Xu L,Wang J,Zhao J,et al.Beauvericin from the endophytic fungus,Fusarium redolens,isolated fromD ioscorea zingiberensisand its antibacterial activity.Nat Prod Commun,2010,5: 811-814.

7 Zhao JL,Xu LJ,Huang YF,et al.Detection of antimicrobial components from extracts of the endophytic fungi associated withParis polyphyllavar.yunnanensisusing TLC-bioautography-MTT assay.Nat Prod ResDev(天然产物研究与开发), 2008,20:28-32.

8 Capasso R,Evidente A,Cutignano A,et al.Fusaric and 9, 10-dehyrofusaric acids and theirmethyl esters fromFusarium nygam ai.Phytochem istry,1996,41:1035-1039.

9 Zonno MC,Vurro M.Inhibition of germination ofO robanche ram osaseeds byFusarium toxins.Phytoparasitica,2002,30: 519-524.

10 AbouzeidMA,Boari A,Zonno MC,et al.Toxicity profiles of potential biocontrol agents ofO robanche ramosa.W eed Sci, 2004,52:6-332.

11 TangJ,TanML,Zhao JL,et al.Detection ofplant antibacterial components by using microplate-MTT colorimetric assay. Nat Prod Res Dev(天然产物研究与开发),2008,20:949-952.

12 Liu H,Wang J,Zhao J,et al.Isoquinoline alkaloids fromM acleaya cordataactive against plant microbial pathogens.Nat Prod Commun,2009,4:1557-1560.

13 KuoMS,Scheffer JM.Evaluation of fusaric acid as a factor in development ofFusarium wilt.Phytopathology,1964,54: 1041-1044.

14 Son S W,K im HY,Choi GJ,et al.Bikaverin and fusaric acid fromFusarium oxysporum show antioomycete activity against Phytophthora infestans.J ApplM icrobiol,2008,104:692-698.

15 Hidaka H,Nagatsu T,Takeya K.Fusaric acid,a hypotensive agent produced by fungi.J Antibiot,1969,22:228-230.