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Bioresource Technology 99 (2008) 89038908

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Bioresource Technology
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Chemical composition and antifungal activity of essential oil and various extract of Silene armeria L.
Vivek K. Bajpai a, Savita Shukla b, Sun Chul Kang a,*
a b

Department of Biotechnology, Daegu University, College of Engineering, Kyoungsan, Kyoungbook 712-714, Republic of Korea P.M.B. Gujrati Science College, Devi Ahilya University, Indore 452-001, MP, India

a r t i c l e

i n f o

a b s t r a c t
The aims of this study were to examine the chemical composition of the essential oil isolated from the oral parts of Silene armeria L. by hydrodistillation, and to test the efcacy of essential oil and various leaf extracts (n-hexane, chloroform, ethyl acetate and methanol) as an antifungal potential. The GCMS analysis determined that 28 compounds, which represented 89.03% of total oil, were present in the oil containing mainly 1-butene, methylcyclopropane, 2-butene and caryophyllene oxide. The oil (1000 ppm/ disc) and the leaf extracts (1500 ppm/disc) revealed remarkable antifungal effect against Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Colletotrichum capsici, Sclerotinia sclerotiorum, Botrytis cinerea and Rhizoctonia solani, in the growth inhibition range of 39.667.6% and 9.361.3%, respectively, along with their respective MIC values ranging from 62.5 to 1000 lg/ml and 125 to 2000 lg/ml. The essential oil had also a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of B. cinerea. Thus, the results obtained in this study demonstrate that S. armeria essential oil and various organic extracts possess a wide range spectrum of fungicidal activity and could become an alternative to synthetic fungicides for controlling certain important plant fungal diseases. 2008 Elsevier Ltd. All rights reserved.

Article history: Received 26 February 2008 Received in revised form 19 April 2008 Accepted 21 April 2008 Available online 5 June 2008 Keywords: Silene armeria Antifungal activity Essential oil Caryophyllene oxide Phytopathogens

1. Introduction The harvest losses due to fungal disease in world crop protection may amount to 12% or even higher in developing countries (Oreke et al., 1994; Agrios, 1997). Many pathogens including Botrytis cinerea (grey mold rot), Fusarium oxysporum (vascular wilt), Sclerotinia sclerotiorum (water soaked spot), Fusarium solani (fruit rot) and Phytophthora capsici (fruit rot) are causing severe damage to agriculture in pre- and post-harvest. Research focused on plant-derived fungicides and their possible application in agriculture is being intensied as these are having enormous potential to inspire and inuence modern agro-chemical research. There is a good reason to suppose that the secondary metabolites of plants have evolved to protect them from attack by microbial pathogens (Benner, 1993). For many years, a variety of different chemical and synthetic compounds have been used as antimicrobial agents to inhibit the plant pathogenic fungi. Antimicrobial chemicals such as benzimidazoles, aromatic hydrocarbons and sterol biosynthesis inhibitors are often used in control of plant disease in agriculture. However, there is a series of problems against the effective use of these chemicals in areas where the fungi have developed resistance (Brent and Hollomon, 1998). In order to overcome this prob* Corresponding author. Tel.: +82 53 850 6553; fax: +82 53 850 6559. E-mail address: sckang@daegu.ac.kr (S.C. Kang). 0960-8524/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.04.060

lem, higher concentrations of these chemicals were used, but this increases the risk of high-level toxic residues in the products. Thus, there has been a growing interest on the research of the possible use of the essential oil and plant extracts, which can be relatively less damaging for pest and disease control in agriculture (Costa et al., 2000). In the past few years, due to concerns regarding the safety of synthetic antimicrobial agents there has been an increase in naturally developed substances, which has resulted in a huge increase in the use of naturally derived compounds such as essential oils and plant extracts as potential antifungal agents. Also, the plants have long been recognized to provide a potential source of chemical compounds or more commonly products, known as phytochemicals including essential oil and plant extracts (Negi et al., 2005). Silene armeria L. known as Catchy (Caryphyllaceae) is a owering plant native to Central and Northern Europe. It is also cultivated and distributed for centuries in temperate regions of Western Asia and America. However, there is no report available on the chemical composition analysis of the essential oil of S. armeria, in general, and its antifungal properties in particular. Hence, efforts have been made, to investigate the role of essential oil and various leaf extracts of S. armeria as an antifungal potential. In the current study, we examined the chemical compositions of the essential oil, isolated from the oral parts of S. armeria and, tested the efcacy of the essential oil and various leaf extracts of

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n-hexane, chloroform, ethyl acetate and methanol against certain important plant pathogenic fungi causing severe destruction to crop, vegetable and ornamental plants.

sclerotiorum KACC 41065, Colletotrichum capsici KACC 410978, Fusarium solani KACC 41092 and Phytophthora capsici KACC 40157. 2.6. Preparation of spore suspension and test samples

2. Methods 2.1. Plant materials The oral parts and the leaves of S. armeria were collected from the local area of Kyoungsan, Republic of Korea, in MayJune 2007 and initially identied by the morphological features and the data base present in the library at the Department of Biotechnology by Prof. Man Kyu Huh. A voucher specimen number (MLDU-0998) has been deposited in the herbarium of College of Engineering, Department of Biotechnology, Daegu University, Republic of Korea. 2.2. Isolation of the essential oil The air-dried plant material (200 g) was subjected to hydrodistillation for 3 h using a modied Clevenger type apparatus. The oil was dried over anhydrous Na2SO4 and preserved in a sealed vial at 4 C until further analysis with a yield of 0.1% (w/w). 2.3. Preparation of leaf extracts The air-dried leaves of S. armeria were pulverized into powdered form. The dried powder (50 g) was extracted with n-hexane, chloroform (CHCl3), ethyl acetate (EtOAc) and 70% methanol (MeOH), separately at room temperature, and the solvents from the combined extracts were evaporated by vacuum rotary evaporator (EYELA N1000). The extraction process yielded in methanol (6.1 g), ethyl acetate (5.6 g), chloroform (5.2 g) and n-hexane (3.5 g) extracts. 2.4. Gas chromatographymass spectrometry (GCMS) analysis The GCMS analysis of the essential oil was performed using a SHIMADZU GCMS (GC-17A) equipped with a ZB-1 MS fused silica capillary column (30 m 0.25 m i.d., lm thickness 0.25 lm). For GCMS detection an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as the carrier gas at a constant ow rate of 1 ml/min. Injector and MS transfer line temperature were set at 220 C and 290 C, respectively. The oven temperature was programmed from 50 C to 150 C at 3 C/min, then held isothermal for 10 min and nally raised to 250 C at 10 C/ min. Diluted samples (1/100, v/v, in methanol) of 1.0 ll were injected manually in the splitless mode. The relative percentage of the oil constituents was expressed as percentages by peak area normalization. Identication of components of the essential oil was assigned by comparison of their retention indices, relative to a series of n-alkane indices on the ZB-1 capillary column and GCMS spectra from the Wiley 6.0 MS data and literature data and whenever possible, by co-injection with authentic compounds (Joulain and Konig, 1998; Adam, 2001). 2.5. Fungal pathogens

The spore suspensions of B. cinerea, F. oxysporum, S. sclerotiorum, C. capsici, F. solani, and P. capsici were obtained from their respective 10 days old cultures, mixed with sterile distilled water to obtain a homogenous spore suspension of 1 108 spore/ml. Essential oil and various leaf extracts (n-hexane, chloroform, ethyl acetate and methanol) of S. armeria were dissolved in 5% dichloromethane and the solvents used for extraction, respectively to prepare the stock solutions with their respective known weights, which were further diluted to prepare test samples, where the nal concentration of the solvent was 0.5% (v/v). 2.7. Antifungal activity assay Petri dishes (9 cm diameter) containing 20 ml of PDA were used for antifungal activity assay, performed on solid media by the disc diffusion method (Duru et al., 2003). Sterile Whatman paper discs of 6 mm diameter were pierced in the agar, equidistant and near the border, where the essential oil 5 ll (1000 ppm/disc) and various leaf extracts of n-hexane, chloroform, ethyl acetate and methanol 7.5 ll (1500 ppm/disc) were used separately. An agar plug of fungal inoculum (6 mm in diameter) was removed from a previous culture of all the fungal strains tested and placed upside down in the center of the petri dishes. The plates were incubated at 25 C for 57 days, until the growth in the control plates reaches the edges of the plates. Growth inhibition of each fungal strain was calculated as the percentage of inhibition of radial growth relative to the control. The plates were used in triplicate for each treatment. The relative growth inhibition of treatment compared to control was calculated by percentage, using the following formula: Inhibition % f1 radial growth of treatmentmm= radial growth of controlmmg 100: 2.8. Determination of minimum inhibitory concentration (MIC) The minimum inhibitory concentrations (MICs) of the essential oil and various leaf extracts of n-hexane, chloroform, ethyl acetate and methanol were determined by twofold dilution method (Murray et al., 1995) against B. cinerea, F. oxysporum, S. sclerotiorum, F. solani, P. capsici and C. capsici. Test samples of essential oil and the extracts (n-hexane, chloroform, ethyl acetate and methanol) were dissolved in the same solvent employed to isolate and extract the essential oil and leaf extracts, respectively. These solutions were serially diluted and were added to PDB (potato dextrose broth) to nal concentrations of 31.25, 62.5, 125, 250, 500, 1000, 2000 and 4000 lg/ml, respectively. A 10 ll spore suspension of each test strains was inoculated in the test tubes in PDB medium and incubated for 27 days at 28 C. The control tubes containing PDB medium were inoculated only with fungal spore suspension. The minimum concentrations at which no visible growth was observed were dened as the MICs, which were expressed in lg/ml. 2.9. Spore germination and growth kinetics assay

The plant pathogenic fungi tested were obtained from the Korean Agricultural Culture Collection (KACC), Suwon, Republic of Korea. Cultures of each fungal species were maintained on potato-dextrose-agar (PDA) slants and stored at 4 C. The fungal species used in the experiment were Botrytis cinerea KACC 40573, Rhizoctonia solani KACC 40111, Fusarium oxysporum KACC 41083, Sclerotinia

Six concentrations of essential oil (31.25, 62.5, 125, 250, 500 and 1000 lg/ml) and one control (0.5% dichloromethane with sterile distilled water) were separately tested for spore germination of different fungi including B. cinerea, F. oxysporum, S. sclerotiorum, C. capsici, F. solani, and P. capsici (Rana et al., 1997). Aliquots of 10 ll

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spore suspension xed with lactophenol-cotton blue from 10-day old cultures of test fungi were placed on both chambers of a hemocytometer by carefully touching the edges of cover slip with the pipette tip and allowed capillary action to ll the counting chamber and observed under the microscope for spore germination. About 200 spores were counted and percentage of spore germination was calculated. All experiments were conducted in triplicate. B. cinerea which appeared to be the more resistant fungus as compared to F. solani to the essential oil in the spore germination study was chosen as test fungus for kinetic study and evaluation of antifungal activity of essential oil. 10 ll spore suspension of this fungal species was inoculated to different concentrations of essential oil (31.25, 62.5 and 125 lg/ml) in a test tube and a homogenous suspension (about 2 105 spore/ml) was made by inverting the test tubes 34 times. After the specic intervals i.e. 30, 60, 90, 120 and 150 min, the reaction mixtures were ltered through Whatman No. 1 lter paper and the retained spores were washed two or three times with sterile distilled water. The lter was then removed and spores were washed off into 10 ml of sterile distilled water. From this 100 ll of spore suspension was taken onto the glass slide and incubated at 24 2 C for 24 h. About 200 spores were counted and percentage spore germination was calculated. Control sets were prepared in 0.5% dichloromethane with sterile distilled water. All experiments were conducted in triplicate. 2.10. Statistical analysis Statistical analysis was performed by using Students t-test. Values are given as standard error of mean (SEM).

Table 1 Chemical composition of volatile oil isolated by hydrodistillation from Silene armeria L. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Total
a b

RIa 443 500 665 785 790 914 930 976 991 1017 1025 1076 1100 1117 1123 1160 1190 1196 1324 1371 1380 1392 1447 1500 1561 1620 1705 1843

Compound 2-Butene 1-Butene Cyclopentane oxide Toluene Methylcyclopropane 2-Butanone 1,8-Nonadiene Pentylfuran b-Myrcene Acetophenone Butanoic acid Linalool Tetrazole Benzyl alcohol 3-Phenylpropanal Benzoic acid Naphthalene Myrtenol Benzene acetic acid cis-Jasmone Eugenol Coumarin a-Humulene Propanoic acid Caryophyllene oxide Pyrrolidine Farnesol Isovaleric acid

%b 17.97 39.20 0.34 0.14 21.48 0.07 0.07 0.09 0.08 0.08 0.05 0.12 0.05 0.08 0.05 0.30 0.08 0.06 0.38 0.10 0.21 0.22 0.07 0.15 7.20 0.21 0.05 0.05 89.03

Retention index relative to n-alkanes on ZB-1 capillary column. Compound percentage.

3. Results 3.1. Chemical composition of essential oil GCMS analyses of the oil led to the identication of 28 different components, representing 89.03% of the total oil. The identied compounds are listed in Table 1 according to their elution order on a ZB-1 capillary column. The oil contained a complex mixture consisting of olenic hydrocarbons, mono- and sesquiterpenes and mono- and sesquiterpene hydrocarbon along with some other essential phytochemicals. The major components in the oil detected were 1-butene (39.2%), methylcyclopropane (21.48%), 2-butene (17.97%) and caryophyllene oxide (7.2%). Terpenoid hydrocarbons were the characteristic constituents of the oil of S. armeria. Coumarin (0.22%), eugenol (0.21%), a-humulene (0.07%), farnesol (0.05%), linalool (0.12%), pentylfuran (0.09%), benzene acetic acid (0.38%), isovaleric acid (0.05%), b-myrcene (0.08%), 2-butanone (0.07%) and acetophenone (0.08%) were also found to be the trace or minor components of S. armeria oil in the present study. 3.2. Antifungal activity of essential oil and crude extracts The oil of S. armeria exhibited a moderate to high antifungal activity against all the plant pathogens tested. As shown in Table 2, essential oil at 5 ll (1000 ppm/disc) showed potent inhibitory effect on the growth of F. oxysporum (54.0%), F. solani (67.6%), P. capsici (59.6%), C. capsici (60.3%), S. sclerotiorum (39.6%), Botrytis cineria (65.6%) and R. solani (58.0%). F. solani, P. capsici, C. capsici and B. cineria were found to be the most inhibited fungal pathogens by the essential oil. Also the leaf extract of methanol at 7.5 ll (1500 ppm/disc) exhibited strong antifungal effect against all the plant pathogens tested, ranging from 19.6% to 61.3%, as compared to chloroform and ethyl acetate extracts (Table 3). Low antifungal effect of hexane extract was observed against only P. capsici and B. cineria with their respective fungal mycelial growth inhibition perTable 2 Antifungal activity of volatile components of the essential oil (5 ll corresponding to 1000 ppm/disc) derived from Silene armeria L. Fungal strain Mycelial growth inhibitiona mm Fusarium oxysporum KACC 41083 Fusarium solani KACC 41092 Phytophthora capsici KACC 40157 Colletotrichum capsici KACC 41078 Sclerotinia sclerotiorum KACC 41065 Botrytis cineria KACC 40573 Rhizoctonia solani KACC 40111 21.0 1.0 14.6 0.5 18.3 1.5 18.0 1.0 27.3 1.1 15.6 0.5 19.0 1.0 % 54.0 2.0 67.6 1.1 59.6 3.5 60.3 2.5 39.6 2.8 65.6 1.1 58.0 2.0 500 125 500 500 1000 62.5 nac MICb

Values are given as mean + S.D. of three experiments. a mm, Radial growth; %, percentage of radial growth inhibition. b Minimum inhibitory concentration (values in lg/ml). c na, Not applicable.

centage of 9.3% and 13.0% (Table 3). Chloroform and ethyl acetate extracts also exhibited signicant antifungal effect against all the plant pathogens tested with their respective fungal mycelial growth inhibition percentage, ranging from 12.3% to 51.6% and 11.6% to 51.6%. One of the fungal pathogens R. solani displayed less susceptibility to the chloroform and ethyl acetate extracts with fungal mycelial growth inhibition percentage of 12.3% and 11.6%, respectively (Table 3). 3.3. Determination of minimum inhibitory concentration (MIC) According to the results given in the Table 2, the minimum inhibitory concentrations (MICs) dened as the lowest concentrations of the essential oil that resulted in complete growth inhibition of F. oxysporum, F. solani, P. capsici, C. capsici, S. sclerotiorum and B. cineria were found to be 500, 125, 500, 500, 1000 and

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Table 3 Antifungal activity of various leaf extracts (7.5 ll corresponding to 1500 ppm/disc) of methanol, hexane, chloroform and ethyl acetate derived from Silene armeria L. Fungal strain Mycelial growth inhibitiona MLextb mm Fusarium oxysporum KACC 41083 Fusarium solani KACC 41092 Phytophthora capsici KACC 40151 Colletotrichum capsici KACC 41078 Sclerotinia sclerotiorum KACC 41065 Botrytis cineria KACC 40573 Rhizoctonia solani KACC 40111 23.0 1.0 17.6 1.1 21.3 0.5 21.3 1.5 36.3 2.1 25.4 0.5 22.4 0.5 % 49.3 2.5 61.3 2.8 53.3 1.1 53.0 3.6 19.6 4.7 44.3 1.1 51.0 1.7 HLextc mm ndf nd 41.0 1.0 nd nd 39.3 1.5 nd % nd nd 9.3 2.5 nd nd 13.0 3.6 nd CLextd mm 25.0 1.0 22.0 1.0 29.6 1.1 28.0 1.0 31.0 1.0 43.3 2.8 39.6 1.1 % 45.0 2.0 51.6 2.5 34.6 2.3 38.0 2.0 31.6 2.5 27.6 0.5 12.3 2.8 ELexte mm 22.0 1.0 23.3 1.1 28.0 1.0 28.3 0.5 31.3 1.5 38.6 1.1 40.0 1.7 % 51.6 2.5 48.6 2.8 38.0 2.0 37.3 1.1 31.0 3.6 11.6 4.0

Values are given as mean S.D. of three experiments. a mm, Radial growth; %, percentage of radial growth inhibition. b MLext, methanol leaf extract. c HLext, hexane leaf extract. d CLext, chloroform leaf extract. e ELext, ethyl acetate leaf extract. f nd, Antifungal activity not detected.

62.5 lg/ml, respectively. F. solani and B. cineria were found to be the most susceptible fungal pathogens to the essential oil of S. armeria. Also the methanol, ethyl acetate and chloroform extracts displayed signicant antifungal effect as minimum inhibitory concentrations against all the plant pathogens tested, with their respective MIC values ranging from 500 to 2000, 125 to 2000 and 500 to 2000 lg/ml (Table 4). As control, the solvent did not effect the growth of the sample strains at the concentration used in this study. No inhibition of any of the fungal pathogens tested was observed using hexane extract as a minimum inhibitory concentration. 3.4. Spore germination and growth kinetics assay The results obtained for essential oil from the spore germination assay of each of the test fungi are shown in Fig. 1. Dichloromethane (0.5%, v/v) as a control did not inhibit the spore germination of any of the plant pathogens tested. There was a signicant inhibition of fungal spore germination by different concentrations of essential oil. A 100% inhibition of fungal spore germination was observed in F. solani and B. cinerea at 62.5 and 125 lg/ml concentrations of essential oil, respectively. Essential oil also exhibited a potent inhibitory effect on the spore germination of F. oxysporum, S. sclerotiorum, P. capsici and C. capsici in the range of 5080% at concentrations ranging from 125 to 500 lg/ml. The antifungal kinetics of the essential oil against B. cinerea is shown in Fig. 2. Exposure of B. cinerea spores to different concentrations of the essential oil for a period of 30150 min caused varyTable 4 Determination of minimum inhibitory concentration (MIC) of various leaf extracts of methanol, hexane, chloroform and ethyl acetate derived from Silene armeria L. Fungal strain MICa MLextb Fusarium oxysporum KACC 41083 Fusarium solani KACC 41092 Phytophthora capsici KACC 40157 Colletotrichum capsici KACC 41078 Sclerotinia sclerotiorum KACC 41065 Botrytis cineria KACC 40573 Rhizoctonia solani KACC 40111
a b c d e f g

ing degree of inhibition of spore germination. An increase in fungicidal activity was observed with increase in exposure time and concentration. The essential oil at 31.25 lg/ml showed antifungal activity but not rapid killing and about 50% inhibition was observed at exposure time of 120 min. However, there was a marked increase in the killing rate at 62.5 and 125 lg/ml after 30 min of exposure, and 95% and 100% inhibition of spore germination was observed on 150 min exposure, respectively. At low concentration, signicant rate of inhibition was the characteristic feature of the essential oil.

4. Discussion The increasing social and economic implications caused by fungi means there is a constant striving to produce safer food crops and to develop new antifungal agents. In general, plant-derived essential oils are considered as non-phytotoxic compounds and potentially effective against plant pathogenic fungi (Pandey et al., 1982). In recent years, interests have been generated in the development of safer antifungal agents such as plant-based essential oils and extracts to control phytopathogens in agriculture (Costa et al., 2000). Historically, many plant oils and extracts have been reported to have antimicrobial properties (Hoffman, 1987; Lawless, 1995). It is important to investigate scientically those plants which have been used in traditional medicines as potential sources of novel antimicrobial compounds (Mitscher et al., 1987). Also, the resurgence of interest in natural control of plant pathogens and increasing consumer demand for effective, safe, natural products means that quantitative data on plant oils and extracts are required. Recently our group and various publications have documented the antifungal activity of essential oils and plant extracts including rosemary, peppermint, bay, basil, tea tree, celery seed and fennel (Bajpai et al., 2007; Morris et al., 1979; Yousef and Tawil, 1980). The hydrodistillation of the oral parts of S. armeria gave light yellowish oil with the major components of the oil having olenic hydrocarbons, mono- and sesquiterpenes and mono- and sesquiterpene hydrocarbons. In recent years, several researchers have reported the mono- and sesquiterpenes, and mono- and sesquiterpene hydrocarbons as the major components of essential oils of plant origin, which have enormous potential to strongly inhibit microbial pathogens (Gudzic et al., 2002; Cakir et al., 2004). In general, the active antimicrobial compounds of essential oils are terpenes, which are phenolic in nature, it would seem reasonable that their antimicrobial or antifungal mode of action might be related to that of other compounds.

HLextc nd nd nd nd nd nd na
g

CLextd 1000 500 1000 2000 2000 2000 na

ELexte 125 500 1000 1000 2000 2000 na

1000 500 500 1000 2000 1000 naf

Minimum inhibitory concentration (values in lg/ml). MLext, methanol leaf extract. HLext, hexane leaf extract. CLext, chloroform leaf extract ELext, ethyl acetate leaf extract. na, Not applicable. nd, Antifungal activity not detected.

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Fig. 1. Effect of different concentrations (lg/ml) of the essential oil of S. armeria on spore germination of tested fungi.

Fig. 2. Kinetics of inhibition of B. cinerea spores by the essential oil of S. armeria.

The essential oil of S. armeria showed remarkable antifungal effect against all the plant pathogens tested. This research work also describes the complex effect of essential oil on fungal spore germination and exhibited a wide range of antifungal activity. During the kinetic study of B. cinerea, it appeared that exposure time of the essential oil had a little effect on the fungicidal activity at lower concentration but at the concentration of 62.5 and 125 lg/ml, the fungicidal action was very rapid and showed 100% spore germination inhibition of B. cinerea. This activity could be attributed to the presence of 2-butene, caryophyllene oxide, methylcyclopropane and a-butylene components present in the oil. On the other hand, one of the fungal pathogen S. sclerotiorum was found slight resistant to the oil at the used concentration with 39.6% of fungal mycelial growth inhibition. This might be ascribed to the existence of mono- and sesquiterpenes in the essential oil as evident by others (Cakir et al., 2004). However, literature from the antifungal search revealed that among the tested plant pathogens monoterpenes were found strongly responsible to increase the growth of certain plant pathogens, these results revealed that there was no signicant correlation between the activity and the percentage of the components identied (Cakir et al., 2004). Also, the results of the antifungal screening showed that various leaf extracts of n-hexane, chloroform, ethyl acetate and methanol derived from S. armeria have potential antifungal activity against the tested plant pathogens. As shown in the Table 2, the essential oil showed remarkable antifungal effect in case of F. solani and B. cineria. Further, methanol, chloroform and ethyl acetate extracts also exhibited signicant amount of fungal mycelium inhibition against the tested plant

pathogens, whereas hexane extract found susceptible only against P. capsici and B. cineria with lower amount of fungal mycelial growth inhibition percentage. This might be attributed to the mode of resistant behavior of the fungi against various substances present in the various solvent extracts. Earlier papers on the analysis and antifungal properties of the essential oils of some species of various genuses have shown that they have a varying degree of growth inhibitory effects against some Fusarium, Botrytis and Rhizoctonia species due to their different chemical compositions (Alvarez-Castellanos et al., 2001; Singh et al., 2002; Bouchra et al., 2003). Bouchra et al. (2003) reported that the oil of seven Moroccan Labiatae, which consists mainly carvacrol, linalyl acetate and tymol as the major components, exhibited a complete mycelial inhibition effect on the growth of B. cinerea. However, the oil of Curcuma longa, which consists mainly of sesquiterpenes and whose major constituents were a-turmerol, b-bisabolene and b-caryophyllene exhibited a complete mycelial growth inhibition against F. oxysporum (Singh et al., 2002). Likewise, lavender and rosemary essential oils were fungitoxic to F. solani, although they had a different chemical composition (Alvarez-Castellanos et al., 2001). On the other hand, the oils of Pistacia vera, P. terebinthus and P. lentiscus had moderate activities at 750 ppm doses against R. solani (Duru et al., 2003). In these essential oils, a-pinene, b-pinene, a-terpineol, bcaryophyllene and terpinen-4-ol were found as major components and they were also characterized in terms of the high contents of monoterpenes (Duru et al., 2003). From some plant oils, such as wintergreen, eucalyptus, clove and sage, there has been much research and reporting of

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V.K. Bajpai et al. / Bioresource Technology 99 (2008) 89038908 Agrios, G.N., 1997. Signicance of plant diseases. In: Plant Pathology, fourth ed. Academic Press, San Diego, pp. 2537. Alvarez-Castellanos, P.P., Bishop, C.D., Pascual-Villalobos, M.J., 2001. Phytochemistry 57, 99102. Bajpai, V.K., Rahman, A., Kang, S.C., 2007. Chemical composition and antifungal properties of the essential oil and crude extracts of Metasequoia glyptostroboides Miki ex Hu. Ind. Crop. Prod. 26, 2835. Benner, J.P., 1993. Pesticidal compounds from higher plants. J. Pestic. Sci. 39, 95 102. Bouchra, C., Achouri, M., Hassani, L.M.I., Hmamouchi, M., 2003. Chemical composition and antifungal activity of essential oils of seven Moroccan Labiatae against Botrytis cinerea Pers. J. Ethnopharmacol. 89, 165169. Brent, K.J., Hollomon, D.W., 1998. Fungicide Resistance: The Assessment of Risk. Monograph No. 2. FRAC, Global Crop Protection Federation, Brussels. pp. 148. Cakir, A., Kordali, S., Zengin, H., Izumi, S., Hirata, T., 2004. Composition and antifungal activity of essential oils isolated from Hypericum hyssopifolium and Hypericum heterophyllum. Flavour Frag. J. 19 (1), 6268. Costa, T.R., Fernandes, F.L.F., Santos, S.C., Oliveria, C.M.A., Liao, L.M., Ferri, P.H., Paulo, J.R., Ferreira, H.D., Sales, B.H.N., Silva, M.R.R., 2000. Antifungal activity of volatile constituents of Eugenia dysenterica leaf oil. J. Ethnopharmcol. 72 (2), 111117. Duru, M.E., Cakir, A., Kordali, S., Zengin, H., Harmandar, M., Izumi, S., Hirata, T., 2003. Chemical composition and antifungal properties of essential oils of three Pistacia species. Fitoterapia 74, 170176. Elad, Y., 1991. Multiple resistance to benzimidazoles dicarboximides and diethofencarb in eld isolates of Botrytis cinerea in Israel. Plant Pathol. 41, 4146. Elad, Y., 1997. Integrated control of foliar disease of green house vegetable crops. In: Hana, A., Achouri, M., Wo, Boudoin (Eds.), Proceeding Symposium International Production et Protection Intgres en Culture Horticole. IAV Agadir, Morocco, p. 518. Gudzic, B., Djokovic, D., Vajs, V., Palic, R., Stojanovic, G., 2002. Composition and antimicrobial activity of the essential oil of Hypericum maculatum Crantz. Flavour Frag. J. 17 (5), 392394. Hoffman, D.L., 1987. The Herb Users Guide. Thomson Publishing Group, Wellingborough, UK. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A.B., 1996. Zulu Medicinal Plants. An Inventory. University of Natal Press, Pietermaritzburg. Joulain, D., Konig, W.A., 1998. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. E. B.-Verlag Hamburg, Hamburg. 1-657. Lawless, J., 1995. The Illustrated Encyclopedia of Essential Oils. Element Books Ltd., Shaftesbury, UK. Marino, M., Bersani, C., Comi, G., 2001. Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceace and Compositae. Int. J. Food Microbiol. 67, 187195. Mitscher, L.A., Drake, S., Gollapudi, S.R., Okwute, S.K., 1987. The society for applied microbiology. J. Appl. Microbiol. 86, 985990. Morris, J.A., Khettry, A., Seitz, E.W., 1979. Antimicrobial activity of aroma chemicals and essential oils. J. Am. Oil Chem. Soc. 56, 595603. Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolke, R.H., 1995. Manual of Clinical Microbiology, sixth ed. ASM, Washington. Negi, P.S., Chauhan, A.S., Sadia, G.A., Rohinishree, Y.S., Ramteke, R.S., 2005. Antioxidant and antibacterial activities of various seabuckthorn (Hippophae rhamnoides L.) seed extracts. Food Chem. 92, 119124. Newall, C.A., Anderson, L.A., Phillipson, J.D., 1996. Herbal Medicines. A Guide for Health-Care Professionals. The Pharmaceutical Press, UK. Oreke, E.C., Dehne, H.W., Schonbeck, F., Weber, A., 1994. Crop Production and Crop Protection: Estimated Loses in Major Food and Cash Crops. Elsevier, Amesterdam. Pandey, D.K., Tripathi, N.N., Tripathi, R.D., Dixit, S.N., 1982. Fungitoxic and phytotoxic properties of the essential oil Caesulia axillaris Roxb. Angew. Bot. 56, 259267. Rana, B.K., Singh, U.P., Taneja, V., 1997. Antifungal activity and kinetics of inhibition by essential oil isolated from leaves of Aegle marmelos. J. Ethanopharmacol. 57, 2934. Reynolds, J.E.F., 1996. Martindale The Extra Pharmacopoeia, 31st ed. Royal Pharmaceutical Society of Great Britain, UK. Singh, G., Singh, O.P., Maurya, S., 2002. Program Crystal Growth and Characterization 45, 7581. Yousef, R.T., Tawil, G.G., 1980. Antimicrobial activity of volatile oils. Die Pharmazie 35, 698701.

toxic and irritant properties (Lawless, 1995; Newall et al., 1996; Reynolds, 1996). In spite of this, most of these oils are available for purchase as whole oils or as a part of pharmaceutical or cosmetic products, indicating that toxic properties do not prohibit their use. However, the ongoing investigation of toxic or irritant properties is imperative, especially when considering any new products for human use, by them medicinal or otherwise. Certain plant extracts and phytochemicals act in many ways on various types of disease complex, and may be applied to the crop in the same way as other agricultural chemicals. S. armeria can also be used as a leading factor in a wide range of activities against many phytopathogens, where these pathogens have developed resistance against the specic fungicides (benzimidazoles, dicarboximides, diethofuncarband and the sterol biosynthesis inhibitors) (Elad, 1991). Among these pathogens, grey mold disease caused by B. cinerea was reported very destructive on crops under greenhouse conditions (Elad, 1997). In our study, it has become clear that both essential oil and leaf extracts of chloroform, ethyl acetate and methanol of S. armeria possesses great potential to strongly inhibit the growth of B. cinerea along with other plant pathogenic fungi tested. In addition, it is also possible that the trace/minor components, such as coumarin, eugenol, a-humulene, farnesol, linalool, pentylfuran, benzene acetic acid, isovaleric acid, b-myrcene, 2-butanone and acetophenone might be involved in some type of antifungal synergism with other active components of essential oil and these ndings are in agreement with previous reports (Marino et al., 2001; Hutchings et al., 1996). In the current study, essential oil and organic extracts showed varying antifungal activities against various plant pathogenic fungi. It would also be interesting to study the effects of essential oil and leaf extracts of S. armeria on medically important fungi for development of new antifungal agents for preventive treatment of serious fungal disease infections in animals and human beings along with plant fungal diseases. In this regard, we have started a program aimed at the evaluation of antifungal activity of essential oil and various leaf extracts (nhexane, chloroform, ethyl acetate and methanol) of S. armeria, in hope to nd out new natural products to be used in the biocontrol of certain important plant pathogenic fungi. In conclusion, S. armeria mediated oil and organic extracts could be applied as alternative industrial products to synthetic fungicides for using in agro-industries and also to screen and develop such novel types of selective and natural fungicides in the biocontrol of many agricultural plant pathogens causing drastic losses to crops. Acknowledgements This work was supported by the RIC program of MOCIE, Republic of Korea. References
Adam, R.P., 2001. Identication of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured Publishing Corporation, Carol stream, IL.

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