식물병연구 Research Article Open Access Res. Plant Dis. 23(3): 268-277 (2017) https://doi.org/10.5423/rpd.2017.23.3.268 Fusarium graminearum Isolation and Characterization of Antifungal Metabolites from Pterocarpus santalinus against Fusarium graminearum Causing Fusarium Head Blight on Wheat *Corresponding author Tel : +82-10-2823-7640 Fax: +82-62-530-2139 E-mail: kjinc@chonnam.ac.kr ORCID https://orcid.org/0000-0003-0753-1966 * Ji-In Kim, Areum Ha, Ae Ran Park, and Jin-Cheol Kim * Department of Agricultural Chemistry, Institute of Environmentally Friendly Agriculture, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Korea Received August 25, 2017 Revised September 5, 2017 Accepted September 5, 2017 Fusarium head bight (FHB) is a devastating disease on major cereal crops worldwide which causes primarily by Fusarium graminearum. Synthetic fungicides are generally used in conventional agriculture to control FHB. Their prolonged usage has led to environmental issues and human health problems. This has prompted interest in developing environmentally friendly biofungicides, including botanical fungicides. In this study, a total 100 plant extracts were tested for antifungal activity against F. graminearum. The crude extract of Pterocarpus santalinus heartwood showed the strongest antifungal activity and contained two antifungal metabolites which were identified as a-cedrol and widdrol by GC-MS analysis. a-cedrol and widdrol isolated from P. santalinus heartwood extract had 31.25 mg/l and 125 mg/l of minimal inhibitory concentration against the spore germination of F. graminearum, and also showed broad spectrum antifungal activities against various plant pathogens. In addition, the wettable powder type formulation of heartwood extract of P. santalinus decreased FHB incidence in dose-dependent manner and suppressed the development of FHB with control values of 87.2% at 250-fold dilution, similar to that of chemical fungicide (92.6% at 2,000-fold dilution). This study suggests that the heartwood extract of P. santalinus could be used as an effective biofungicide for the control of FHB. Keywords: Pterocarpus santalinus, Fusarium graminearum, Fusarium head blight, a-cedrol, widdrol Fusarium graminearum,, Research in Plant Disease pissn 1598-2262, eissn 2233-9191 www.online-rpd.org, (Desjardins, 2006; Goswami, 2004; McMullen, 1997). 1963 40-60%, 80-100% (Chung, The Korean Society of Plant Pathology This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Research in Plant Disease Vol. 23 No. 3 269 1975), 10. 74, 90, 98,,, 69-84% (Ryu, 2011). 2002, 07, 10 11 (Park, 2012; Shim, 2009). trichothecenes zearalenone, trichothecene deoxynivalenol, (Peraica Domijan, 2001). metconazole, propiconazole, prothioconazole, tebuconazole demethylation inhibitor (DMI)., (Bradley, 2009; 2010), (Bondalapati Stein, 2010) (Ibáñez-Vea, 2011). 9, (Son, 2011). DMI 30 50% (McMullen, 2012). DMI (Erysiphe graminis), (Monilinia fructicola), (Mycosphaerella graminicola) DMI (Delye, 1998; Leroux, 2007; Luo Schnabel, 2008; Yin, 2009)., terpenoids, alkaloids, flavonones, polyketides,,, (Copping Duke, 2007; Copping Menn, 2000; Le Dang, 2012). ( : Santali Lignum Rubrum) (Pterocarpus santalinus),,.,, benzofurans (Nagaraju, 1991; Wu, 2011). Candida albicans, Aspergillus niger, Rhizopus, Fusarium, F. graminearum (Jyothi Chaitanya, 2014; Vandita, 2013). 100 F. graminearum ( ).,,.... (,, ) 100 ethanol (EtOH) F. graminearum. ( ).. F. graminearum
270 Research in Plant Disease Vol. 23 No. 3 F. graminearum Z-3639 (Bowden Leslie, 1999), potato dextrose agar (PDA; Becton, Dickinson and Company, MD, USA). carboxymethylcellulose (CMC, carboxymethylcellulose 15 g, NH 4 NO 3 1 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 0.5 g, yeast extract 1 g, 1 l) (Cappellini Peterson 1965; Leslie Summerell, 2008). F. graminearum 20% glycerol -70 o C.. (200 g) methanol (MeOH, 3 l). MeOH, residue 3 l MeOH. rotary evaporator (OSB-2100, EYELA, Japan) 45 o C. MeOH (305 g) 70% MeOH n-hexane 2. 70% MeOH rotary evaporator MeOH ethyl acetate (EtOAc) 2. EtOAc anhydrous sodium sulfate. butanol (BuOH) 2 BuOH. 4 F. graminearum, n-hexane,. F. graminearum n- hexane (3 g) silica gel column (3.6 cm i.d. 60 cm, Kiesel gel 60, 70-230 mesh, 200 g; Merck, Darmstadt, Germany) n-hexane:acetone (9:1, v/v). silica gel 60 F 254 glass plate (Merck, Germany) n-hexane:acetone (9:1, v/v) thin-layer chromatography (TLC) TLC 5 (F1-F5). F. graminearum, F4 F5 silica gel column chromatography (2.75 g) 4 (F41-F44), F41 (909.7 mg) column chromatography. F41 silica gel column (2.8 cm i.d. 60 cm, Kiesel gel 60, 230-400 mesh, 50 g; Merck, Germany) EtOAc:n-hexane (1:7, v/v) 1 (99 mg). F3 (342 mg) TLC (Kiesel gel 60, 0.5 mm film ; Merck, Germany). toluene:etoac:acetic acid (60:20:1, v/v/v), 2 (13 mg). gas chromatography-mass spectrometry (GC-MS). GC-MS (QP2010; Shimadzu, Kyoto, Japan) DB-5 column (0.25 mm i.d. 60 m, 0.25 mm film, Agilent Technologies, Palo Alto, CA, USA) ion source 200 o C, injector 250 o C oven 100 o C 2 280 o C 10 o C 280 o C 3.. 100 EtOH, 4 (n-hexane, EtOAc, BuOH, ) 2 F. graminearum macroconidia. CMC F. graminearum 4 25 o C 200 rpm 4 4 o C 10,000 rpm 15., potato dextrose broth (PDB; Becton, Dickinson and Company). hemocytometer (0.0025 mm 2, Marienfeld Superior, Lauda-Königshofen, Germany) 1 10 6 macroconidia/ml PDB. 48 well plate well 198 l, 2 l, EtOH 2,000, 1,000, 500 mg/l, 4 2,000, 1,000, 500, 250, 125, 62.5, 31.25, 15.625 mg/l, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91 mg/l. EtOH EtOH, n-hexane EtOAc acetone, BuOH MeOH, 10% EtOH, dimethylsulfoxide(dmso). (acetone, MeOH, DMSO) 1%.
Research in Plant Disease Vol. 23 No. 3 271 3, 25 o C (6 ). well 10 l (100 ; Harris Swift M1000-D; Swift Optical Instruments, Schertz, TX, USA).. 3 well 300.. n-hexane 1, 2 F. graminearum (Z-3639) Rhizoctonia solani (,, ), Magnaporthe oryzae (KACC46522, ), Fusarium oxysporum f.sp. lycopersici (KACC40043), Scleotinia homoeocarpa ( ), Colletotrichum coccodes ( ), Cryphonectria parasitica ( ). DMSO. PDB agar plug 25 o C 7 4 50 mg /ml. 96 well plate well 99 l 1 l 500, 250, 125, 62.5, 31.25, 15.63 mg/l 25 o C.. 1% DMSO, 3 2. (MIC: minimum inhibitory concentration).... n-hexane (2 g) acetone white carbon (1.5 g) acetone 2. sodium dodecyl sulfate (CR-SES, 0.5 g) sodium poly (naphthalene formaldehyde) sulfonate (CR-WP100, 0.5 g), Kaoline 5.5 g (Yoon, 2010)., 500 250. 24 F. graminearum 2 10 5 conidia/ml. (20 cm i.d. 8 cm). 2 10. 3 30. difenoconazole propiconazole 13% (Syngenta, ), (Kim, 2016).. (A: FHB index, B: FHB index, FHB index = /100, (% Disease severity) = 100 ( / )). F. graminearum. 100 F. graminearum (data not shown)., Candida albicans Aspergillus niger, Lasiodiploidia, Rhizopus (Vandita, 2013)., Pterocarpus indicus MeOH Trycomyces palustris Coriolus versicolor, P. indicus flvaonoids F. oxysporum, Cochliobolus miyabeanus, Trichoderma harzinum (Kusuma, 2005; Pilotti, 1995).,,, (Kondeti, 2010; Kwon, 2006;
272 Research in Plant Disease Vol. 23 No. 3 Manjunatha, 2006). MeOH n-hexane, EtOAc, BuOH,, n-hexane EtOAc, 250 mg/l 500 mg/l MIC Fig. 1 n-hexane EtOAc, BuOH F. graminearum. BuOH 2000 mg/l 100%,. Fig. 1. Effects of n-hexane extract, ethyl acetate, butanol, and aqueous layers fractionated from Pterocarpus santalinus on the spore germination of Fusarium graminearum. Each value represents mean±standard deviation of two runs with three replicates. One hundred spores were observed using light microscopy for each treatment with three times. Different letters on bars indicate significant differences according to Duncan s multiple range test at P=0.05.. 4 n-hexane TLC F. graminearum. GC-MS (Fig. 2A, 3A), total ion chromatogram (TIC) Fig. 2. GC-MS total ion chromatogram (A) and electron-impact mass spectrum (B) of compound 1 isolated from the heartwood of Pterocarpus santalinus.
Research in Plant Disease Vol. 23 No. 3 273 Fig. 3. GC-MS total ion chromatogram (A) and electron-impact mass spectrum (B) of compound 2 isolated from the heartwood of Pterocarpus santalinus.. (Electron impact-mass spectrum) library search (Fig. 2B, 3B), 1 a-cedrol, 2 widdrol. 222, C 15 H 26 O., bioautography assay EtOAc, n-hexane a-cedrol widdrol (data not shown). F. graminearum a-cedrol widdrol n-hexane EtOAc. a-cedrol cedrol, (+)-cedrol 8- -H-cedran-8-ol,, Cupressus Juniperus (cedar oil) sesquiterpene alchol (El- Sawi, 2007; Ismail, 2013). Widdrol Juniperus chinensis (Jin, 2015),. isoflavonoid, terpenoid, lignin (Arunakumara, 2011), a-cedrol widdrol Pterocarpus a-cedrol widdrol. F. graminearum. a-cedrol widdrol F. graminearum,, a-cedrol MIC 31.25 mg/l, widdrol 62.5 mg/l a-cedrol (Fig. 4). Fig. 5A 6, Fig. 5B 5C a-cedrol widdrol 62.5 mg/l.,. a-cedrol widdrol Pterocarpus
274 Research in Plant Disease Vol. 23 No. 3 diyne -8,9,10 -triol. F. graminearum MIC 125 62.5 mg/l. (acedrol MIC, 31.25 mg/l; widdrol MIC, 62.5 mg/l) polyacetylene. Fig. 4. Effects of a-cedrol and widdrol isolated from Pterocarpus santalinus on the spore germination of Fusarium graminearum. Each value represents mean±standard deviation of two runs with three replicates. One hundred spores were observed using light microscopy for each treatment with three times. Juniperus. Tumen (2012) Juniperus a-cedrol, thujopsene widdrol,., Nuñez (2006) Juniperus widdrol B. cinerea, Guleria (2008) Thuja orientalis a-cedrol Alternaria alternata. F. graminearum a-cedrol widdrol Pterocarpus a- cedrol widdrol. Kim (2016) F. graminearum ciryneol C 1-heptadecene-11,13-. a-cedrol widdrol, F. graminearum., 7 (Table 1). a- cedrol widdrol, M. oryzae a-cedrol widdrol., a-cedrol B. cinerea 7.81 mg/l MIC S. homoeocarpa C. coccodes, E. parasitica 31.25 mg/l MIC. Widdrol M. oryzae B. cinerea 31.25 mg/l. F. graminearum a-cedrol wid- Table 1. Inhibition of a-cedrol and widdrol isolated from Pterocarpus santalinus against mycelial growth of plant pathogenic fungi in vitro Fungal species a-cedrol MIC (mg/l) Rhizoctonia solani 62.5 125 Fusarium graminearum 250 500 Widdrol Magnaporthe oryzae 125 31.25 Sclerotinia homoeocarpa 31.25 125 Colletotrichum coccodes 31.25 62.5 Endothia parasitica 31.25 125 Botrytis cinerea 7.8125 31.25 Fig. 5. Influence of a-cedrol and widdrol isolated from Pterocarpus santalinus on germination of Fusarium graminearum spores. Microscopic observation was performed 6 h after chemical treatment to the spores of F. graminearum. (A) control, (B) a-cedrol (62.5 mg/l), (C) widdrol (62.5 mg/l).
Research in Plant Disease Vol. 23 No. 3 275 Table 2. Disease control efficacy of the wettable powder type formulation of n-hexane layer of Pterocarpus santalinus against Fusarium head blight on wheat under field condition a Treatment drol 250 mg/l 500 mg/l MIC. F. graminearum.. n-hexane (PS-WP20). PS-WP20 500 250, 47.0% 87.2%, 4000 2000 84.2% 92.6% (Table 2).. Dilution Disease incidence (%) Control value (%) Control - 64.4±15.8 - PS-WP20 b 250 24.1±10.3 87.2a PS-WP20 500 44.9±3.3 47.0b Almuri c 2000 23.3±5.8 92.6a Almuri 4000 32.2±6.9 84.2ab a The wheat seedlings were inoculated with spore suspension of Fusarium graminearum 24 hours after treatment of chemical fungicide or PS-WP20 which were sprayed to run-off on the leaves. Each value represents mean±standard deviation of three replicates. Means with the same capital letter in FHB index are not significantly different (P<0.05) according to Duncan s multiple range test. b PS-WP20, wettable powder type formulation of the n-hexane extract of P. santalinus. c Dilution ( 2,000) of Almuri is compatibilizer concentration.,,,.,,, (Kondeti, 2010; Kwon, 2006; Manjunatha, 2006). flavonoids, terpenoids, phenolic compounds, alkaloids, saponins, tannins glycosides (Kondeti, 2010; Narayan, 2005), a-cedrol widdrol. a-cedrol, (Sabine, 1975)., widdrol. F. graminearum sesquitepene, a- cedrol widdrol,. n-hexane. a-cedrol, a-cedrol 15.8% 19.0% cedar oil. F. graminearum,.,,. Fusarium graminearum... 100 F. graminearum., GC-MS a-cedrol widdrol. a-cedrol widdrol F. graminearum 31.25 mg/l 62.5 mg/l, F. graminearum.,, 250 2,000 (92.6%)
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