Antifungal activity of Araliae Continentalis Radix extract on rice sheath blight

*Corresponding author: Myoung-Jun Jang, Department of Plant Resources, Kongju National University, College of Industrial Sciences, Yesan, Republic of Korea, South Korea. Mobile: +82-41-330-1200, Fax: +82-41-330-1209. E-mail: plant119@kongju.ac.kr Received: 09 December 2015; Revised: 18 September 2016; Accepted: 21 September 2016; Published Online: 30 October 2016 Oh, et al.: Antifungal activity of Araliae Continentalis Emir. J. Food Agric ● Vol 28 ● Issue 11 ● 2016 819 Radix have been reported as a substance with high antifungal activity recently (Han, 2004). To this end, this clinical study examined the source material and investigated the use of Araliae Continentalis Radix for the development of an eco-friendly antifungal agent for rice sheath blight. MATERIALS AND METHODS Extraction of the antifungal substance We purchased Araliae Continentalis Radix locally grown from an oriental medicinal material vendor located in Dang Jin Si, Chung Cheong Nam Do, Korea and used it as the test material. The raw materials were crushed with a grinding mill (HMF-34 0, Hanil, Korea) and then used as the sample for the extraction. Ethanol was used as the extracting solvent. We mixed the powder specimen of Araliae Continentalis Radix and ethanol at a ratio of 1:5 (w/v) and performed an agitated extraction at room temperature for 24 hours. The extracted material was filtered with filtration paper (Whatman, No.2) followed by decompressive concentration (Vacuum Filtration) in a water bath at 45°C in a rotary evaporator (N-1000, Eyela, Japan). Then, we suspended the Araliae Continentalis Radix extract by adding 1 L of distilled water and fractionated the extract with chloroform followed by ethyl acetate then n-butanol and finally water obtaining 5.51 g of chloroform, 4.24 g of ethyl acetate, 7.85 g of n-butanol, and 10.92 g of water through inspissation. After decompressive concentration of the ethyl acetate layer which showed a strong inhibition activity, we performed silica gel column chromatography with the solvents CHCl3-MeOH (100:1 to 1:2) and divided it into 6 fractions in order to isolate the antifungal substance. Among those 6 fractions, the No. 5 fraction showed a strong antifungal activity. Therefore, to isolate the active substance from the 5th fraction, we performed silica gel column chromatography with the solvents hexane-acetone (20:1 to 10:1) and obtained compounds 1 (130 mg) and 2 (20 mg). Then, these compounds were identified as ent-Pimara-8(14),15-diene-19-oic acid and ent-Kaur-16-en-19-oic acid through MS and NMR data analyses(supplemental data). Antifungal activity of the Araliae Continentalis Radix extract We used Rhizoctonia solan Kuhn AG-1(IA)(KACC: 40106) supplied by the KACC (Korea Agricultural Culture Collection) as a publicly announced strain of rice sheath blight. To investigate the antifungal activity of the Araliae Continentalis Radix extract, we first mixed PDA media with the extract for final concentrations of 62.5, 125, 250 and 500 mg. L-1 before the media hardened when the media was made. Then, we put 10 ml each of the mixture into 9 cm-diameter petri dishes, and after checking for solidification, we took a mycelia disc (5 mm diameter) of sheath blight and placed it onto the center of the medium. The mycelia was cultured at 25°C, and the mycelia growth rate was measured to determine the antifungal activity of the Araliae Continentalis Radix extract. As a control, we used Hexaconazole Emulsion after mixing it with the Araliae Continentalis Radix extract at the same concentration and compared the antifungal actions using the same method mentioned above. Antifungal activity assay of ent-pimara-8(14),15diene-19-oic acid and ent-Kaur-16-en-19-oic acid After cultivating Rhizoctonia solan Kuhn AG-1(IA), the concentration of the mycelia (1×104 piece/mL) was confirmed with a Haematocytometer. Then, the plate medium (test sample) was prepared by smearing the cultured Rhizoctonia solan Kuhn AG-1(IA) onto the PDA medium with a spread stick. The ent-Pimara-8(14),15diene-19-oic acid and ent-Kaur-16-en-19-oic acid were prepared so that their final concentrations were 62.5, 125, 250, and 500 mg. L-1. Each acid was absorbed onto a 8 mm disc paper, and the disc papers were completely dried. The dried discs were placed onto the plates containing the PDA medium and the Rhizoctonia solan Kuhn AG-1(IA) and incubated in a 25°C incubator. The antifungal activity for each concentration of the acids was compared by measuring the clear zone generated around the disc. Test of antifungal control on rice sheath blight for packaging The testing for packaging fungicide control for rice sheath blight was implemented at the Disease and Insect Pests Observational Plot of the Agricultural Technology Center in Seocheon-gun, Choong Chung Nam-do on August 1, 2013. The selected specimen was Saenuri. A foliar spray of the fungicide was done twice on August 1 and August 7 for each concentration (62.5, 125 and 250mg. L-1) of the Araliae Continentalis Radix extract. Disease and insect pest observational plot As for control, after spraying a Hexaconazole emulsion at the same levels, we calculated the damage level in accordance with the Disease and Insect Pest Investigation Standards of rice sheath blight on August 14, 2013 based on the research, investigation and analysis criteria for agricultural science technology. The damage rate was calculated as follows: Damage Rate (%) = (3n1+ 2n2+ 1n3/3 N) × 100, where N is the number of invested tiller, n2 the number of diseased tiller up to the leaves, n1 the number of diseased Oh, et al.: Antifungal activity of Araliae Continentalis 820 Emir. J. Food Agric ● Vol 28 ● Issue 11 ● 2016 tiller up to the branch leaves, and n3 the number of diseased tiller (NDT) up to the third leaves. All statistical analyses were done with the Duncan test using SAS 8.0 (Statistical Analysis System). RESULTS AND DISCUSSION Antifungal activity of the Araliae Continentalis Radix extract on Rhizoctonia solan Kuhn AG-1(IA) Araliae Continentalis Radix was reported to have antifungal effects on pathogenic microbes and some plant pathogenic fungi(Oh et al., 2013) in their study reported that Araliae Continentalis Radix extract has a high level of antifungal activity against Pyricularia grisea, and Han (2004) also reported that Araliae Continentalis Radix Extract has an antimicrobial activity against gram-positive bacilli. Because Araliae Continentalis Radix is known to have antimicrobial and antifungal activities, we tested the antifungal activity of the Araliae Continentalis Radix Extract against Rhizoctonia solan Kuhn AG-1(IA), and the results are shown in Table 1 and Fig. 1. As for the untreated petri plates, the hyphae of the sheath blight grew to the extent that they almost covered the entire surface of the 9 cm petri dish; however, in the petri dish in which the control and extract were mixed together, the growth level of the hyphae was notably lower compared to the untreated petri plates. For the control, the hyphae of the sheath blight could not grow at all, and the diameter of the hyphae was10 mm for every treatment concentration. The hyphae had a growth diameter of 17 mm when the extract concentration was 125 mg. L-1 which was the smallest diameter of growth. Moreover, at extract concentrations of 500 mg. L-1 and 250 mg. L-1, the growth diameters of the hyphae were 18 mm and 21 mm respectively, showing no large difference. In addition, at the low extract concentration level of 62.5 mg. L-1, the growth diameter was 24 mm. The results show that the extract has antifungal activities against sheath blight (Table 1). From the aforementioned results, it was possible to confirm that Araliae Continentalis Radix has an antifungal activity against sheath blight which was also confirmed by extracting ent-Pimara-8(14),15-diene-19-oic acid and ent-Kaur-16-en-19-oic acid from the Araliae Continentalis Radix extract. ent-Pimara-8(14), 15-diene-19-oic acid and ent-Kaur-16-en-19-oic acid have already been reported to have a high level of antimicrobial activity against gram-positive bacillus(Han et al., 2004), but its antifungal activity against sheath blight has not been reported until now. When we checked the number of colonies that formed after smearing Rhizoctonia solan Kuhn AG-1(IA) onto the medium, the untreated petri plate had so many colonies to the extent that accurate counting of the colonies was impossible, while the control showed such a high level of antifungal activity to the extent that it was not possible to check any of the colonies. ent-Pimara-8(14), 15-diene-19-oic acid and ent-Kaur-16en-19-oic acid extracted from the Araliae Continentalis Radix extract had a lower level of antifungal activity than that of the Hexaconazole emulsion, which was used as a control, but still had a higher antifungal activity than the untreated petri plate (Table 2). Table 1: Effectiveness of the antifungal activity of the Araliae Continentalis Radix extract at various concentrations against Rhizoctonia solan Kuhn AG-1(IA) Treatment (mg.L-1) Growth diameter of hypha (mm)