BioControl (2009) 54:697–707 DOI 10.1007/s10526-009-9220-2
Antimycotic activities of Cinnamon-derived compounds against Rhizoctonia solani in vitro Van-Nam Nguyen Æ Dang-Minh-Chanh Nguyen Æ Dong-Jun Seo Æ Ro-Dong Park Æ Woo-Jin Jung
Received: 3 November 2008 / Accepted: 9 April 2009 / Published online: 28 April 2009 Ó International Organization for Biological Control (IOBC) 2009
Abstract In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was
Handling Editor: Reijo Karjalainen. V.-N. Nguyen R.-D. Park Glucosamine Saccharide Materials-National Research Laboratory, Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University, Gwangju 500-757, South Korea V.-N. Nguyen e-mail:
[email protected]
exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%. Keywords Antifungal activity Cinnamomum cassia Plant extracts Rhizoctoni solani
R.-D. Park e-mail:
[email protected] D.-M.-C. Nguyen D.-J. Seo W.-J. Jung (&) Environment-Friendly Agriculture Research Center (EFARC), Division of Applied Bioscience and Biotechnology, Institute of Agriculture Science Technology, Chonnam National University, Gwangju 500-757, South Korea e-mail:
[email protected] D.-M.-C. Nguyen e-mail:
[email protected] D.-J. Seo e-mail:
[email protected]
Introduction The control of plant diseases depends primarily upon the application of chemical fungicides. However, these substances have the potential to exert toxic effects on humans and wildlife as well as to cause environment pollution (Knight et al. 1997). Therefore, alternative methods for the biological control of plant diseases have been developed, including the use
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of enzymes, chitinases, proteases, and b-1,3-glucanases (Jung et al. 2002; Nguyen et al. 2007). Antibiotics have also been utilized for the biological control of plant diseases. Because plants produce a wide variety of physiologically active substances, they often serve as alternative sources of chemicals for the treatment of plant diseases (Slusarenko et al. 2008). Medicinal plants have been used in traditional medicine as diuretics, topical anti-inflammants, and haemostatics (Burt 2004). Medicinal plants usually produce many natural products, such as phenols, flavonoids, quinons, tannins, alkaloids, saponins, sterols, and volatile essential oils. These secondary metabolites have various functions, including antimicrobial, insecticide and appetite suppressant properties (Akhtar et al. 2008; Isman 2000; Liu and Zhang 2004; Mares et al. 2005; Soylu et al. 2006; Yazaki et al. 2008; Wilson et al. 1997). Additionally, natural products are generally easily biodegradable. Therefore, they do not tend to persist in the environment (Akhtar et al. 2008). Increasing interest in environmentally friendly sustainable agriculture and horticulture as organic farming has resulted in increased demand for pesticides produced by plants. As a result, the use of natural products for the control of fungal diseases in plants is considered to be an interesting alternative to synthetic fungicides, and such compounds may soon represent a new class of safer disease control agents. Indeed, some phytochemicals of plant origin, such as azadirachtin, carvone, and pyrethroids, have already been formulated as botanical pesticides and used successfully in integrated pest management programs (Shmutterer 1990). In addition, the extracts of specific plants are traditionally used as natural fungicides in small scale farming systems in which the use of synthetic chemicals is not economically feasible (Tegegne and Pretorius 2007). Essential oils obtained from aromatic plants such as oregano, thyme, lavender, rosemary, fennel, and laurel have been shown to completely inhibit the growth of Phytophthora infestans, as well as to induce considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shriveling and protoplast leakage (Soylu et al. 2006). Furthermore, cytomorphological alterations of the hyphae of Fusarium solani, Rhizoctonia solani, Pythium ultimum var. ultimum, and Colletotrichum lindemuthianum was observed when these fungi were treated with garlic extracts (Bianchi et al.
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1997). Many studies have been conducted to identify novel anti-fungal materials produced by plants over the past few years. Such studies have focused on identifying compounds with broad-spectrum activity that could be useful for the treatment of plant diseases and have resulted in the isolation of many antimicrobial compounds (Chu et al. 2006; Fiori et al. 2000). However, the use of properly developed and scientifically tested plant products for the control of plant diseases is currently limited (Tegegne and Pretorius 2007). Cinnamon has long been used for its biter-tinic and aromatic properties. Recently, the water extract of Cinnamon was found to inhibit the germination of Oidium murrayae spores (Chu et al. 2006), and the essential oil of Cinnamon has been reported to exert nematicidal activity (Choi et al. 2006). In this study, we focused on (1) initial assessment of the antifungal activities of methanolic extracts from medicinal plants in vitro against the following phytopathogenic fungi: R. solani, C. gloeosporioides, F. solani, P. capsici, and B. cinera and (2) partial purification and estimation of antifungal substrate extracted from Cinnamon against R. solani in vitro.
Materials and methods Chemicals and materials The following solvents were used in this study: n-hexane, chloroform, ethyl acetate, and n-butanol. All solvents were of reagent grade and purchased from Junsei Chemical Company Ltd (Japan). Silica gel 60 F254 plates were purchased from Merck (Germany). The phytopathogenic fungi, Phytophthora capsici KACC 40157, R. solani KACC 40146, F. solani KACC 40384, C. gloeosporioides ATCC 32097, and Botrytis cinera KCTC 6973, were obtained from the Korea Research Institute of Bioscience and Biotechnology, South Korea.
Compound extracts from medicinal plants About 26 locally available plants used in traditional medicine in Vietnam were collected and identified (Pham, 1993). The leaves, stems, roots, bulbs, and flowers were then cut into 3 cm pieces and placed in
Antimycotic activities of Cinnamon-derived compounds
paper bags and dried by heating in oven at 50°C for three days and stored at room temperature. The plant preparations were then extracted in 99% methanol at a ratio of 1:5 (v/v, dry plant material/solvent) at 30°C with shaking at 150 rpm for seven days. The extracts were vacuum filtered through a Whatman No 2 filter and the solvents were dried by vacuum evaporation at 40°C to give a dried paste. The pastes were then weighed and redissolved in methanol to give a paste with a final concentration of 10%. Antifungal activity of plant extracts Five fungal strains, P. capsici, R. solani, F. solani, C. gloeosporioides, and B. cinera, were used to screen the antifungal activity of the plant extracts. About 6-mm-diameter mycelial culture block of the same age were cut and then transferred to the center of a new Petri plate containing potato dextrose agar medium. The fungal preparation was incubated at 25°C for one day (R. solani), three days (P. capsici, C. gloeosporioides, and B. cinera) and four days (F. solani) until the fungal colony reached to 3 cm in diameter. To screen for antifungal activity, 8-mm diameter paper discs were placed so that their edge was 10 mm from the edge of the hyphal colony. About 30 ll of the 10% plant extract were applied to the paper discs and the samples were then incubated at 25°C. Then, the antifungal activity of the plant extracts was estimated based on the distance from the colony edge to the edge of the paper discs. Strong inhibition (???) was estimated as a distance of [5 mm, average inhibition (??) was considered to be a distance of 2–5 mm, slight inhibition (?) was considered to be a distance of 1–2 mm and no inhibitory activity (-) was considered to be a distance\1 cm. The distances of the treatments were determined when hyphal mycelia in control (treated with 30 ll of methanol) reached at the edge of the paper discs. Partial fractionation of antifungal substances from Cinnamon extract The initial isolation of active compounds from the liquid–liquid portion of the extract paste (20 g) dissolved in water was sequentially partitioned into n-hexane, chloroform, ethyl acetate, and n-butanol three times. Equal volumes of each solvent and extract solution were then mixed by shaking for 3 min in a
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separation funnel. The organic portions of the solvents were then combined and dried by rotary evaporation at 40°C (Jang et al. 2001). The pastes were redissolved in methanol to give a concentration of 10%. These samples were then used for the antifungal assay and further purification of the active compound. Antifungal activity of the Cinnamon compound isolated by partial fractionation To estimate the inhibition rate of the solvent fractions against R. solani, 30 ll of compounds with different concentrations (0% as a control or 1, 2.5, and 5%) were placed on paper discs, and the mycelial growth was then measured at 10-h intervals. Growth values were converted into inhibition percentages according to the following formula: IR (%) = ((GC–GT)/ GC) 9 100, where IR is the inhibition rate, and GC and GT represent mycelial growth in the control and treatment, respectively (Soylu et al. 2006). Separation of the active substances from the n-hexane solvent fraction The solvent-fractionized compounds were spotted on silica gel plates and then separated in developing solvent comprised different ratios of n-hexane:ethyl acetate, after which they were observed under a UV light at 254 nm using CAMAG Video Scan, CH-4143 Mutten 1 (Switzerland). The best separation of compounds was observed when a 80:20 (v/v) solvent ratio of hexane:ethyl acetate was used. For initial purification of the active compounds, paste containing the Cinnamon compound from the n-hexane fraction at a concentration of 5% was continuously separated on silica gel plates (Wang et al. 2004). Briefly, the paste solution was streaked onto two Silica gel plates, after which the compounds were separated in the hexane:ethyl acetate (80:20, v/v) developing solvent. The samples were then observed under UV light at a wavelength of 253 nm and the four separated compound regions were identified and marked. Each region of the silica gel plates was then removed and eluted in methanol. The methanol solution was then evaporated, after which the paste was redissolved in methanol to give a solution with a final concentration of 10%, which was utilized for further study of the antifungal activity and HPLC analysis.
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Antifungal activity of TLC-separated Cinnamon compounds To determine the mycelial growth rate in response to treatment with the compounds fractions purified by TLC, 30 ll of each compound were applied on paper discs at a concentration of 0% (methanol alone) as a control, 0.5% (150 lg), 1% (300 lg) and 2% (600 lg). The discs were then applied to plates containing the fungal samples and mycelial growth was measured at 5, 10, 20 and 30 h after treatment. The mycelial growth rate of R. solani was then calculated based on the following formula: GR = GRT/GRC, where GRT is the mycelial growth observed in the treatments and GRC is the mycelial growth observed in the control. Hyphal morphology of R. solani treated with Cinnamon compound fraction I To determine if hyphae were damaged in response to treatment with compound fraction I (Comp. I), the R. solani mycelia growing in the PDA medium were treated with the compounds by direct contact. To accomplish this, paper discs containing Comp. I were placed directly on young hyphae at the edges of the colony and on old hyphae inside the colony. After 1 h of treatment, the fungal blocks were cut from the control area (just methanol) and the treated area and then stained with lactophenol blue (Dhingra and Sinclair 2000) and observed by light microscopy (Soylu et al. 2006). Statistical analysis Data were compared using Tukey’s Studentized Range (HSD) test, with a p B 0.05 being taken to indicate statistical significance. All data were analyzed using the Statistical Analysis System 9.1 (SAS institute, 2004) and are presented as the mean value ± standard deviation.
Results Antifungal activity of plant methanolic extracts against phytopathogenic fungi The extract pastes from medicinal plants were redissolved in methanol and characteristics of each
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extract such as smell, color and residue in the solvent were recorded (data not shown). The compound solutions were then stored at 4°C until used. The fungicidal activity of the 26 medicinal plant extracts against five important phytopathogenic fungi, P. capsici KACC 40157, R. solani KACC 40146, F. solani KACC 40384, C. gloeosporioides ATCC 32097, and B. cinera KCTC 6973 are shown in Table 1. Out of the 26 plant extracts, six extracts from Alpinia galanga (L) Willd, Artemisia capillaris Thunb, Cinnamomum cassia Blume, Glycyrrhiza uralensis Fisch, Senecio aureus Pursh, and Zingiber officinale Roscoe showed inhibitory activity against mycelial growth. However, only two plant extracts from A. galanga and G. uralensis showed inhibitory activity against all five fungi, whereas the remainder of the extracts showed inhibitory activity towards only some of the fungi tested. Overall, six plant extracts showed slight or moderate mycelial inhibition, with the extract from Cinnamon showing the highest fungicidal activity against R. solani. Therefore, the Cinnamon extract was subjected to further purification and subsequent testing against R. solani in vitro. Plant extracts obtained from A. galanga, C. cassia, G. uralensis and Z. officinale exerted inhibition against R. solani. Therefore, the mycelial inhibition of R. solani exerted by different concentrations of these plant extracts was tested. The results revealed that the mycelial inhibition exerted by the four plant extracts occurred in a dose-dependent manner. The mycelial growth of R. solani gradually decreased as the dose of paste in methanol increased from 0 to 3 mg (Fig. 1). In addition, the development of hyphae was found to slow in response to treatments. However, none of the four crude plant extracts completely inhibited hyphal growth of R. solani.
Antifungal activity of the fractionations of Cinnamon extract against R. solani A dual solvent system was used to separate the antifungal substances in the Cinnamon extract. To accomplish this, the methanolic extract (20 g) was dissolved in water and then sequentially partitioned into n-hexane, chloroform, ethyl acetate and n-butanol. The pastes of the organic solvent portions (n-hexane (1.59 g), chloroform (0.75 g), ethyl acetate (0.82 g) and n-butanol (4.11 g)) were then obtained
Antimycotic activities of Cinnamon-derived compounds
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Table 1 Antifungal activity of crude methanolic extracts from medicinal plants against five important plant pathogens by paper disc assay in vitro Name of plant species
Plant parts tested
Phytopathogenic fungus Phy
Rhi
Fus
Col
Bot
Albizia lebbeck
Seed
-
-
-
-
-
Allium fistulosium
Seed
-
-
-
-
-
Alpinia galanga
Bulb
?
??
??
?
??
Artemisia capillaris
Leaf
-
-
-
?
-
Aster sp.
Seed
-
-
-
-
-
Azadirachta indica
Seed
-
-
-
-
-
Cassia glauca
Plant
-
-
-
-
-
Cassia angustifolia
Leaf
-
-
-
-
Chrysanthemum sp.
Flower
-
-
-
-
-
Cinnamomum cassia
Bark
-
???
?
-
-
Cymbopogon nardus Ganoderma lucidum
Leaf Root
-
-
-
-
-
Glycyrrhiza uralensis
Bulb
??
?
?
?
?
Leucaena glauca
Seed
-
-
-
-
-
Leucaena glauca
Leaf
-
-
-
-
-
Lonicera japonica
Root
-
-
-
-
-
Luffa cylindrica
Fruit
-
-
-
-
-
Mentha spicata
Leaf
-
-
-
-
-
Mentha gracilis
Leaf
-
-
-
-
-
Mimosa pudica
Flower
-
-
-
-
-
Phyllanthus urinaria
Plant
-
-
-
-
-
Perilla ocymoides
Leaf
-
-
-
-
-
Polygonum multiflorum
Root
-
-
-
-
-
Senecio aureus
Leaf
?
-
-
?
-
Tagetes erecta
Plant
-
-
-
-
-
Zingiber officinale
Bulb
-
??
?
?
-
Phy: Phytophthora capsici (KACC 40157), Rhi: Rhizoctonia solani (KACC 40146), Fus: Fusarium solani (KACC 40384), Col: Colletotrichum gloeosporioides (ATCC 32097), Bot: Botrytis cinera (KCTC 6973). -: no inhibitory activity, ?: slight inhibitory, ??: medium inhibitory activity, and ???: high inhibitory activity
by rotary evaporation at 40°C, while the water portion of the extracts was freeze-dried. The four paste fractions were then redissolved in methanol to give 10% substrate concentration. Solution of n-hexane and chloroform fractions was oily, slight yellow in color while solution of ethyl acetate and n-butanol fractions was soluble in water and red color. The antifungal activity of compounds against R. solani from the four solvent fractions was then preliminarily screened. For initial identification of the antifungal compounds, the methanolic crude extract and fractions of the four solvent: n-hexane, chloroform, ethyl acetate,
and n-butanol, were assayed for mycelial inhibition of R. solani on PDA medium by paper disc assay. A wide range of inhibitory activity against mycelial growth was observed in response to treatment with the n-hexane, chloroform, methanolic crude, ethyl acetate and n-butanol fractions (Table 2). The differences of inhibitory activity were statistically significant (P B 0.05) in range of n-hexane, chloroform, crude extract, ethyl acetate, and n-butanol at 10 and 30 h after treatment. The highest antifungal activity with significant difference (P B 0.05) was at 2.5% paste concentration of n-hexane fraction at 10 and 30 h after treatment.
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Fig. 1 Inhibition of Rhizoctonia solani mycelial growth by crude methanolic extracts from Zingiber officinale (A), Cinnamomum cassia (B), Glycyrrhiza glabra (C), and Albinia galangal (D). Plant crude methanolic extracts were placed on paper discs, which were subsequently used to treat the fungi. The discs contained either methanol alone (control) (1) or methanol solution containing 0.75 mg (2), 1.5 mg (3) or 3 mg of the pastes (4)
There were statistically significant reductions in the mycelial growth of R. solani when the fungus was treated with the pastes containing the n-hexane and the chloroform fractions at concentrations of 2.5 and 5% (P B 0.05). Indeed, when R. solani was treated with paste containing the n-hexane fraction at a concentration of 5%, the inhibition rate reached 100% at 10 h after treatment; however, no significant difference was shown at 2.5 and 5% paste concentration of n-hexane fraction. Very low inhibition rate was observed in response to treatment with the n-butanol fractions, and there was highly significant difference of inhibition between n-hexane and n-butanol treatments over time of treatment at P B 0.05. Separation and initial purification of the antifungal substances The products obtained from the four solvent fractions and the crude extract were spotted onto silica gel
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plates and then separated in a gradient of hexane– ethyl acetate with different ratios (100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 by volume). Among the solvent ratios evaluated, the mixture of n-hexane and ethyl acetate (80:20, v/v) showed clearly separated compounds on silica gel plates. Based on UV light analysis (253 nm), six major colored substances with the following Rf values were identified in the n-hexane fraction: 0.76, 0.62, 0.56, 0.41, 0.31 and 0.13 (Fig. 2). The n-hexane fraction containing all of the compounds identified by TLC exerted the highest antifungal activity. Therefore it was chosen for initial purification of the active compounds. The 5% paste was chromatographed on two silica gel plates with a mobile phase of hexane–ethyl acetate (80:20, v/v). Four major compound fractions with Rf values of 0.76, 0.62, 0.41 and 0.13, which are hereafter referred to as compound fraction I (Comp. I), compound fraction II (Comp. II), compound fraction III (Comp. III) and compound fraction IV (Comp. IV), were cut
Antimycotic activities of Cinnamon-derived compounds Table 2 Percentage of Rhizoctonia solani mycelial growth inhibition induced by different concentrations of Cinnamon compounds separated by organic solvents
Solvent
n-Hexane
Chloroform
Crude extract Values given in the column are the mean ± standard deviation based on three replicates. Values in the column that are followed by the same letter are not significantly different (P B 0.05) as determined by Tukey’s Studentized Range (HSD) test
Compound concentration (%)
At 10 h
At 30 h
100.0 ± 0.0a
2.5
a
94.5 ± 4.7
100.0 ± 0.0a 98.5 ± 0.2a
1.0 5.0
65.5 ± 11.0 89.6 ± 5.8a
b
59.4 ± 4.0b 67.3 ± 7.4b
2.5
56.5 ± 6.9bc
55.9 ± 3.0b
1.0
45.0 ± 1.6
cd
30.3 ± 8.4c
38.5 ± 3.3
de
14.5 ± 2.3de
26.8 ± 1.9
ef
11.4 ± 2.6de
1.0
8.5 ± 1.5
gh
7.0 ± 3.3de
5.0
21.2 ± 1.6fg
18.1 ± 1.7cd
2.5
19.1 ± 1.6
fg
11.4 ± 1.8de
8.4 ± 1.8
gh
3.7 ± 0.9e
2.4 ± 1.7
h
12.0 ± 1.6de
0.6 ± 0.1
h
8.7 ± 1.2de
0.6 ± 0.1
h
7.3 ± 2.0de
5.0
1.0 n-Butanol
Mycelial inhibition after treatment (% ±SD)
5.0
2.5 Ethyl acetate
703
5.0 2.5 1.0
from the silica gel plates and then eluted in methanol at 30°C with shaking at 150 rpm for 24 h. The compound solutions were then filtered with Whatman paper No 2, after which the solvent was evaporated to
give 110, 70, 50, and 40 mg of Comp. I, Comp. II, Comp. III and Comp. IV, respectively. These compounds were then dissolved in methanol to give a final concentration of 10%, and were then assayed for antifungal activity against R. solani. Antifungal activity and change in hyphal morphology in response to treatment with the TLC-separated compounds
Fig. 2 TLC separation of compounds from different solvent fractions of Cinnamon extract: crude compound in methanol (1), n-hexane fraction (2), ethyl acetate fraction (3), chloroform fraction (4), and n-butanol fraction (5). Major compounds (Comp. I, Comp. II, Comp. III, Comp. IV) were separated using a mobile phase comprised hexane:ethyl acetate (80:20, v/v) and detected at 253 nm UV light
Data regarding the inhibition of mycelial growth in response to the compounds derived from TLC are shown in Fig. 3. The antifungal activity against R. solani in response to treatment with Comp. I and Comp. II was greater than that of Comp. III and Comp. IV. In addition, mycelial formation by R. solani was found to decrease gradually in response to increasing doses of the compounds from 0 to 600 lg. The time course analysis of the mycelial growth rate in response to treatment with 300 and 600 lg of the pastes are described in Fig. 4. The mycelial growth rate was reduced slowly in response to treatment with Comp. III and Comp. IV until 30 h after treatment, after which the growth rate slowly recovered. Conversely, the mycelial growth rate was greatly reduced to 0.51 and 0.51 in response to treatment with 300 lg of Comp. I and Comp II, respectively, and to 0.19 and 0.18 in response to
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Fig. 3 Mycelial growth inhibition by Cinnamonderived compounds against Rhizoctonia solani KACC 40146 at 30 h after treatment. Comp. I (A), Comp. II (B), Comp. III (C), and Comp. IV (D) were separated by silica gel plates and then redissolved in methanol. About 30 ll of each compound solution containing either methanol alone as a control (1), or 150 lg (2), 300 lg (3), or 600 lg paste in methanol (4) were placed on paper discs
treatment with 600 lg of Comp. I and Comp II, respectively, at 20 h after treatment. Finally, the growth rate recovered 30 h after treatment with 300 lg of Comp. I and Comp. II, but did not recover after treatment with 600 lg. Microscopic observation of R. solani hyphae exposed to Comp. I revealed degenerative changes in the hyphal morphology when compared with the hyphae of the controls (Fig. 5). Specifically, 1 h after contact exposure of the hyphae to Comp. I, changes in the mycelial morphology were clearly visible. For example, the hyphae were degraded and contained cells with no cytoplasm or depleted levels of cytoplasm when R. solani was treated with 0.5% paste. Seriously damaged hyphae were observed in response to treatment with Comp. I at a concentration of 1 or 2%, while shrinkage of the hyphae was observed in response to treatment with paste that contained Comp. I at a concentration of 1% and swollen hyphal tips, collapsed hyphae, and completely destroyed hyphae were observed in response
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to treatment with paste that contained Comp. I at a concentration of 2%.
Discussion The results of the present study demonstrated that six plants exerted antifungal activity against phytopathogenic fungi (Table 1). Out of the plant extracts, A. galanga and G. urionalensis showed inhibitory activity toward five fungi in this experiment. However, the plant extracts showed only moderately or slight inhibitory activity when evaluated by paper disc assay. Cinnamon extract paste dissolved in methanol was found to be highly effective against the growth of R. solani in vitro when applied as a paper disc assay or by direct contact (Fig. 1). When applied as a paper disc assay, the R. solani hyphal tips were swollen and the cytoplasm in the young hyphal cells was clear when compared with the control. Furthermore, after
Antimycotic activities of Cinnamon-derived compounds
Fig. 4 Time course of the growth inhibition of Rhizoctonia solani induced by the TLC-separated compounds, 300 lg of each compound (A) and 600 lg of each compound (B), values given are the mean ± standard deviation of three replicates
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two-day treatment with low concentrations of the plant extract, hyphal growth tents were recovered and continuously developed. When the plant extract was applied by direct contact, the hyphae were completely destroyed. Specifically, the cell walls were deformed and collapsed, which resulted in the death of the hyphae in response to treatment with paste containing the Cinnamon extract at the concentration of 1%. However, the older hyphae of R. solani were relatively unaffected by treatment with the Cinnamon extract. These data should be considered when applying Cinnamon extract to control diseases in plants. Various plant extracts or essential oils have been reported to exert different levels of antifungal activity in vitro against phytopathogenic fungi, including oregano (Origanum syriacum var. bevanii), thyme (Thymbra spicata subsp. spicata), lavender (Lavandula stoechas subsp. stoechas), rosemary (Rosmarinus officinalis), fennel (Foeniculum vulgare) and laurel (Laurus nobilis) against P. infestans (Soylu et al. 2006) and essential oils of Achillea millefolium, Cymbopogon citratus, Eucalyptus citriodora, and Ageratum conyzoides against the fungus Didymella bryoniae (Fiori et al. 2000).
Fig. 5 Effects of fungicidal Comp. I on the hyphal morphology of Rhizoctonia solani as observed microscopically (at 409 magnification). Healthy hyphae growing on control plates (A), lightly affected hyphae with a transparent cytoplasm in samples treated with 0.5% Comp. I (B), shrunken hyphae in samples treated with 1% Comp. I (C), and swollen and abnormal hyphal tips (solid arrow) and collapsed hyphae (dashed arrow) in samples treated with 2% Comp. I (D) for 1 h
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In this study, the antifungal substances from the methanolic extract of Cinnamon were initially purified by solvent fractionation using silica gel plates for a short time, and the purified compound showed a high antifungal activity toward R. solani (Fig. 3). These results are supported by the results of other studies in which Cinnamon oil compounds were reported to exert activity against Dermatophagoides farinae and D. pteronyssinus when applied as fumigants (Kim et al. 2008), as well as antifungal activity toward Oidium murrayae (Chu et al. 2006) and nematicidal activity against the pine wood nematode, Bursaphelenchus xylophilus (Choi et al. 2006). Mycelial development of R. solani was strongly inhibited in response to treatment with paste containing Comp. I at a concentration of 1% (Fig. 4). In addition, treatment with Comp. I at a concentration of 1 or 2% via direct contact resulted in the collapse or complete destruction of hyphae (Fig. 5). Such inhibitors are believed to block the synthesis of chitin in fungal cell walls (Liu and Zhang 2004). Based on these results, Cinnamon methanol extracts and their substrate fractions could be useful as fungicide against R. solani. Further studies of extracts described here should be conducted to purify active compounds and to elucidate the chemical formula. The antifungal activity of the compounds against R. solani could be applied on the plants in the field. Acknowledgments This work was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea, and by the Korea Science and Engineering Foundation (KOSEF) through the National Research Lab program funded by the Ministry of Science and Technology (No. 1030000032206J0000-32210).
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