식물병연구 Research Article Open Access Res. Plant Dis. 23(3): (2017) Fusarium graminearum Isolation and

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1 식물병연구 Research Article Open Access Res. Plant Dis. 23(3): (2017) Fusarium graminearum Isolation and Characterization of Antifungal Metabolites from Pterocarpus santalinus against Fusarium graminearum Causing Fusarium Head Blight on Wheat *Corresponding author Tel : Fax: kjinc@chonnam.ac.kr ORCID * 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 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 , eissn (Desjardins, 2006; Goswami, 2004; McMullen, 1997) %, % (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 ( by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Research in Plant Disease Vol. 23 No ), , 90, 98,,, 69-84% (Ryu, 2011). 2002, 07, (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

3 270 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, 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, 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 o C 10 o C 280 o C 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 ( mm 2, Marienfeld Superior, Lauda-Königshofen, Germany) 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, 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%.

4 Research in Plant Disease Vol. 23 No , 25 o C (6 ). well 10 l (100 ; Harris Swift M1000-D; Swift Optical Instruments, Schertz, TX, USA).. 3 well 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 mg /ml. 96 well plate well 99 l 1 l 500, 250, 125, 62.5, 31.25, 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)., F. graminearum conidia/ml. (20 cm i.d. 8 cm) 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;

5 272 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= 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.

6 Research in Plant Disease Vol. 23 No 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 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.

7 274 Research in Plant Disease Vol. 23 No. 3 diyne -8,9,10 -triol. F. graminearum MIC mg/l. (acedrol MIC, 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 mg/l MIC. Widdrol M. oryzae B. cinerea 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 Fusarium graminearum Widdrol Magnaporthe oryzae Sclerotinia homoeocarpa Colletotrichum coccodes Endothia parasitica Botrytis cinerea 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).

8 Research in Plant Disease Vol. 23 No 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-WP , 47.0% 87.2%, % 92.6% (Table 2).. Dilution Disease incidence (%) Control value (%) Control ± PS-WP20 b ± a PS-WP ± b Almuri c ± a Almuri ± ab 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 F. graminearum., GC-MS a-cedrol widdrol. a-cedrol widdrol F. graminearum mg/l 62.5 mg/l, F. graminearum.,, 250 2,000 (92.6%)

9 276 Research in Plant Disease Vol. 23 No %.. Conflicts of Interest The authors declare that they have no competing and commercial interests in this work. Acknowledgement This work was supported by the Cooperative Research Program for Agricultural Science and Technology Development (Project PJ ), Rural Development Administration, Republic of Korea. References Arunakumara, K. K. I. U., Walpola, B. C., Subasinghe, S. and Yoon, M.-H Pterocarpus santalinus Linn. f. (Rath handun): a review of its botany, uses, phytochemistry and pharmacology. J. Korean Soc. Appl. Biol. Chem. 54: Bondalapati, K. and Stein, J Validation of barley DON risk prediction model. In: Proc 2010 National Fusarium Head Blight Forum, eds. by S. Canty, A. Clark, A. Anderson-Scully, D. Ellis and D. Van Sanford, pp Milwaukee, WI. Bowden, R. L. and Leslie, J. F Sexual recombination in Gibberella zeae. Phytopathology 89: Bradley, C. A., Adee, E. A., Ebelhar, S. A, Grybauskas, A. P., Hollingsworth, C. R., Kirk, W. W., McMullen, M. P., Milus, E. A., Osborne, L. E., Ruden, K. R. and Young, B. G Application timings of Caramba and Prosaro foliar fungicides for management of FHB and DON. In: Proc 2009 National Fusarium Head Blight Forum, eds. by S. Canty, A. Clark, J. Mundell, E. Walton, D. Ellis and D. Van Sanford, 34 pp. Orlando, FL. Bradley, C., Adee, E., Ebelhar, S., Dill-Macky, R., Wiersma, J., Grybauskas, A., Krik, W., McMullen, M., Halley, S. and Milus, E Multistate uniform fungicide evaluations for control of Fusarium head blight and associated mycotoxins. In: Proc 2010 National Fusarium Head Blight Forum, eds. by S. Canty, A. Clark, A. Anderson-Scully, D. Ellis and D. Van Sanford, 74 pp. Milwaukee, WI. Cappellini, R. and Peterson, J Macroconidium formation in submerged cultures by a non-sporulating strain of Gibberella zeae. Mycologia 57: Chung, H. S Cereal scab causing mycotoxicoses in Korea and present status of mycotoxin researches. Kor. J. Mycol. 3: Copping, L. G. and Duke, S. O Natural products that have been used commercially as crop protection agents. Pest Manag. Sci. 63: Copping, L. G. and Menn, J. J Biopesticides: a review of their action, applications and efficacy. Pest Manag. Sci. 56: Delye, C., Bousset, L. and Corio-Costet, M. F PCR cloning and detection of point mutations in the eburicol 14a-demethylase (CYP51) gene from Erysiphe graminis f. sp. hordei, a recalcitrant fungus. Curr. Genet. 34: Desjardins, A. E Fusarium Mycotoxins: Chemistry, Geneticsand Biology. APS Press, St. Paul, MN. El-Sawi, S. A., Motawae, H. M. and Ali, A. M Chemical composition, cytotoxic activity and antimicrobial activity of essential oils of leaves and berries of Juniperus phoenicea L. grown in Egypt. Afr. J. Tradit. Complement. Altern. Med. 4: Goswami, R. S. and Kistler, H. C Heading for disaster: Fusarium graminearum on cereal crops. Mol. Plant Pathol. 5: Guleria, S., Kumar, A. and Tiku, A. K Chemical composition and fungitoxic activity of essential oil of Thuja orientalis L. grown in the north-western Himalaya. Z. Naturforsch. C 63: Ibáñez-Vea, M., Lizarraga, E., González-Peñas, E. and Cerain, A. L Co-occurrence of type-a and type-b trichothecenes in barley from a northern region of Spain. Food Control 25: Ismail, A., Lamia, H., Mohsen, H., Samia, G. and Bassem, J Chemical composition, bio-herbicidal and antifungal activities of essential oils isolated from Tunisian common cypress (Cupressus sempervirens L.). J. Med. Plants Res. 7: Jin, S., Yun, H. J., Jeong, H. Y., Oh, Y. N., Park, H. J., Yun, S.-G., Kim, B. W. and Kwon, H. J Widdrol, a sesquiterpene isolated from Juniperus chinensis, inhibits angiogenesis by targeting vascular endothelial growth factor receptor 2 signaling. Oncol. Rep. 34: Jyothi Chaitanya, P., Chandrashekar, R. and Lakshmi Bhavani, N Assessment of histochemical, phytochemical and antimicrobial activity of Pterocarpus santalinus L wood extracts. European J. Biomed. Pharm. Sci. 1: Kim, J.-I., Kim, K., Park, A. R., Choi, G. J., Park, H. W., Kim, I. S. and Kim, J.-C Biological control of Fusarium head blight on wheat by polyacetylenes derived from Cirsium japonicum roots. Res. Plant Dis. 22: (In Korean) Kondeti, V. K., Badri, K. R., Maddirala, D. R., Thur, S. K. M., Fatima, S. S., Kasetti, R. B. and Rao, C. A Effect of Pterocarpus santalinus bark, on blood glucose, serum lipids, plasma insulin and hepatic carbohydrate metabolic enzymes in streptozotocin-induced diabetic rats. Food Chem. Toxicol. 48: Kusuma, I. W., Itoh, K. and Tachibana, S Antifungal activities against plant pathogenic fungi of flavonoids isolated from Amboyna wood. Pak. J. Biol. Sci. 8: Kwon, H., Hong, Y., Kim, K., Han, C., Cho, S., Choi, J. and Kim, B.-W Methanolic extract of Pterocarpus santalinus induces apoptosis in HeLa cells. J. Ethnopharmacol. 105: Le Dang, Q., Lim, C.-H. and Kim, J.-C Current status of botanical pesticides for crop protection. Res. Plant Dis. 18: Leroux, P., Albertini, C., Gautier, A., Gredt, M. and Walker, A. S

10 Research in Plant Disease Vol. 23 No Mutations in the CYP51 gene correlated with changes in sensitivity to sterol 14a-demethylation inhibitors in field isolates of Mycosphaerella graminicola. Pest Manag. Sci. 63: Leslie, J. F. and Summerell, B. A The Fusarium Laboratory Manual. John Wiley & Sons. Luo, C. X. and Schnabel, G The cytochrome P450 lanosterol 14a-demethylase gene is a demethylation inhibitor fungicide resistance determinant in Monilinia fructicola field isolates from Georgia. Appl. Environ. Microbiol. 74: Manjunatha, B Hepatoprotective activity of Pterocarpus santalinus L. f., an endangered medicinal plant. Indian. J. Pharmacol. 38: McMullen, M., Jones, R. and Gallenberg, D Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Dis. 81: McMullen, M., Bergstrom, G., De Wolf, E., Dill-Macky, R., Hershman, D., Shaner, G. and Van Sanford, D A unified effort to fight an enemy of wheat and barley: Fusarium head blight. Plant Dis. 96: Nagaraju, N., Prasad, M., Gopalakrishna, G. and Rao, K Blood sugar lowering effect of Pterocarpus santalinus (Red Sanders) wood extract in different rat models. Int. J. Pharmacogn. 29: Narayan, S., Devi, R., Srinivasan, P. and Devi, C Pterocarpus santalinus: a traditional herbal drug as a protectant against ibuprofen induced gastric ulcers. Phytother. Res. 19: Nuñez, Y. O., Salabarria, I. S., Collado, I. G. and Hernández-Galán, R The antifungal activity of widdrol and its biotransformation by Colletotrichum gloeosporioides (penz.) Penz. & Sacc. and Botrytis cinerea Pers.: Fr. J. Agric. Food Chem. 54: Park, J.-M., Shin, S.-H., Kang, C.-S., Kim, K.-H., Cho, K.-M., Choi, J.-S., Kim, H.-M. and Park, J.-C Fungicide effects in vitro and in field trials on Fusarium head blight of wheat. Res. Plant Dis. 18: (In Korean) Peraica, M. and Domijan, A.-M Contamination of food with mycotoxins and human health. Arh. Hig. Rada Toksikol. 52: Pilotti, C. A., Kondo, R., Shimizu, K. and Sakai, K An examination of the anti-fungal components in the heartwood extracts of Pterocarpus indicus. Mokuzai Gakkaishi 41: Ryu, J.-G., Lee, S.-H., Son, S.-W., Lee, S.-H., Nam, Y.-J., Kim, M.-J., Lee, T. and Yun, J.-C Natural occurrence of Fusarium head blight and its mycotoxins in 2010-harvested barley and wheat grains in Korea. Res. Plant Dis. 17: (In Korean) Sabine, J. R Exposure to an environment containing the aromatic red cedar, Juniperus virginiana: procarcinogenic, enzymeinducing and insecticidal effects. Toxicology 5: Shim, H. G., Noh, T.-H., Lee, D.-K., Song, J.-J. and Oh, I.-S Occurrence of rice headblight in Honam area and infectionmechanism. In: Annual Report for 2008, pp National Institute of Crop Science. Son, S.-W., Nam, Y.-J., Lee, S.-H., Lee, S.-M., Lee, S.-H., Kim, M.-J., Lee, T., Yun, J.-C. and Ryu, J.-G Toxigenic fungal contaminants in the 2009-harvested rice and its milling-by products samples collected from rice processing complexes in Korea. Res. Plant Dis. 17: (In Korean) Tumen, I., Eller, F. J., Clausen, C. A. and Teel, J. A Antifungal activity of heartwood extracts from three Juniperus species. Bio- Resources 8: Vandita, P., Amin, N., Khyati, P. and Monisha, K Effect of phytochemical constituents of Ricinus communis, Pterocarpus santalinus, Terminalia belerica on antibacterial, antifungal and cytotoxic activity. Int. J. Toxicol. Pharmacol. Res. 5: Wu, S.-F., Chang, F.-R., Wang, S.-Y., Hwang, T.-L., Lee, C.-L., Chen, S.-L., Wu, C.-C. and Wu, Y.-C Anti-inflammatory and cytotoxic neoflavonoids and benzofurans from Pterocarpus santalinus. J. Nat. Prod. 74: Yin, Y., Liu, X., Li, B. and Ma, Z Characterization of sterol demethylation inhibitor-resistant isolates of Fusarium asiaticum and F. graminearum collected from wheat in China. Phytopathology 99: Yoon, M.-Y., Cha, B. and Kim, J.-C Recent trends in studies on botanical fungicides in agriculture. Plant Pathol. J. 29: 1-9. Yoon, M. Y., Choi, G., Choi, Y., Jang, K., Park, M., Cha, B. and Kim, J. C Effect of polyacetylenic acids from Prunella vulgaris on various plant pathogens. Lett. Appl. Microbiol. 51:

212 Research in Plant Disease Vol. 20 No. 3., 7 (Kim, 2004). Colletotrichum (Park Kim, 1992),.. (Agrios, 2008)... 1,202, 256 92, 147, 11 (RDA, 2014).

212 Research in Plant Disease Vol. 20 No. 3., 7 (Kim, 2004). Colletotrichum (Park Kim, 1992),.. (Agrios, 2008)... 1,202, 256 92, 147, 11 (RDA, 2014). 식물병연구 Research Article Open Access Res. Plant Dis. 20(3) : 211-215(2014) http://dx.doi.org/10.5423/rpd.2014.20.3.211 Protective Activity of the Mixtures of Pine Oil and Copper Hydroxide against Bacterial