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Short communication
Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro Q1
Dong-Jun Seo a, Dang-Minh-Chanh Nguyen a, b, Ro-Dong Park a, Woo-Jin Jung a, * a Division of Applied Bioscience and Biotechnology, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Science, Chonnam National University, Gwangju 500-757, Republic of Korea b Western Highlands Agriculture Forestry Science Institute, 53 Nguyen Luong Bang Street, Buon Ma Thuot, Vietnam
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 October 2013 Received in revised form 25 December 2013 Accepted 30 December 2013 Available online xxx
A novel chitosanecinnamon bead carrier was prepared in this study. Chitosan was mixed with cinnamon powder (CP) and cinnamon extract (CE) to obtain chitosanecinnamon powder (CCP) beads and chitosan ecinnamon extracted (CCE) beads, respectively. The potential antifungal and nematicidal activities of CCP and CCE were investigated against Rhizoctonia solani and Meloidogyne incognita in vitro. Relative antifungal activity of the CCP (5% CP) bead-treated R. solani was 30.9 and 23.9% after 1 and 2 day incubations, respectively. Relative antifungal activity of the CCE (0.5% CE) bead-treated R. solani was 4.3, 3.0 and 4.2% after 1, 2 and 3 days of incubation. Inhibition of hatch by CCP beads with CP of 5% was 78.8%. Inhibition of hatch by CCE beads with CE of 0.5% was 82.0%. J2 mortality following the CCP (5% CP) and CCE (0.5% CE) bead treatments was 85.0 and 95.8%, respectively against M. incognita after 48 h incubations. Ó 2014 Published by Elsevier Ltd.
Keywords: Chitosan beads Cinnamomum cassia Antifungal activity Nematicidal activity
1. Introduction Chitosan is comprised of linear high molecular weight polyglucosamine chains and possesses distinct chemical and biological properties. Therefore, chitosan has been used as a swollen bead support for preparing immobilized enzymes and conjugate drug carriers [1e3]. Cinnamon (Cinnamomum cassia) is a tall (10e20 m) native medicinal plant that grows in the Central Highlands of Vietnam. The tree bark is harvested throughout the year, preferably in summer and autumn. It is applied as therapy for dyspepsia, colic, diarrhoea, cholera, dysentery, coryza, influenza, cough, asthma, paresis, and snake-bites [4]. Additionally, cinnamon extracts are known for their antifungal activity and are used in bakery products for their aromatic properties. Nielsen and Rios [5] demonstrated that cinnamon extracts exert antifungal effects through their volatile components, and reported that they have a potential application in active packaging. Previous studies have suggested that crude cinnamon extracts exert nematicidal activity against the pine wood nematode, Bursaphelenchus xylophilus [6]. Thus, methanol extracts of cinnamon and their sub-fractions could be useful as fungicides against Rhizoctonia solani [7]. Additionally, Nguyen et al. [8] showed that antioxidant enzyme activity such as superoxide dismutase,
* Corresponding author. Tel.: þ82 62 530 3960, fax: þ82 62 530 2139. E-mail address: [email protected] (W.-J. Jung).
catalase, and ascorbate peroxidase increase in the leaves of cucumber plants treated with 5 or 10 mg/mL C. cassia extract compared with leaves of untreated plants on day 28 after treatment. These findings suggest that the induction of pathogenesisrelated proteins following C. cassia extract treatment may strengthen the plants against various biotic stressors. Furthermore, cinnamyl acetate purified from cinnamon shows high nematicidal activity against Meloidogyne incognita [9]. In this study, chitosan beads were used as carriers to immobilize the cinnamon activities. Applying cinnamon to swollen chitosan beads has not been extensively studied. This study was designed to investigate the activities of cinnamon combined with swollen chitosan beads against the phytopathogens R. solani and M. incognita in vitro. 2. Materials and methods 2.1. Materials Chitosan powder [90% deacetylation, 10 cPs (in 0.5% acetic acid þ 0.5% chitosan solution, at 20 C)] was purchased from Keumho Chemical Co., Ltd. (Seoul, Korea). C. cassia bark was purchased from Buon Ma Thuot market, a medicinal plant and herb shop in DakLak province, Vietnam. R. solani KACC 40111 was obtained from the Korea Agricultural Culture Collection (Suwon, Korea). M. incognita eggs and second-stage juveniles (J2) were
0882-4010/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.micpath.2013.12.007
Please cite this article in press as: Seo D-J, et al., Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2013.12.007
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extracted from infected cucumber roots in a growth chamber. All other chemicals were of analytical grade. 2.2. Preparation of chitosanecinnamon powder (CCP) and chitosanecinnamon extract (CCE) beads The cinnamon bark was cut into 0.5e1.0 cm pieces and powdered using a mortar and pestle to obtain the cinnamon powder (CP). The cinnamon bark was cut into 2e3 cm pieces and extracted with 99% methanol at a ratio of 1:5 (v/v, dry bark/methanol) at 30 C with shaking at 150 rpm for 7 days. The filtrate was evaporated to dryness under a vacuum at 40 C (Eyela N-1000, Tokyo, Japan) and then lyophilized to obtain the cinnamon extract (CE). The CCP was prepared by dissolving chitosan powder (7 g) in 100 mL of 7% acetic acid and then adding it to 0, 1, 3, and 5 g CP, respectively. Similarly, the CCE was prepared by dissolving chitosan powder (7 g) in 100 mL of 7% acetic acid and then 0, 0.1, 0.3, and 0.5 g of CE were added. The resulting viscous solution of CCP and CCE was de-gassed under vacuum and dropped into 200 mL of an alkali coagulating solution (H2OeMeOHeNaOH ¼ 4:5:1, w/w/w) to prepare highly swollen 3.5 mm average diameter spherical beads [10]. The CCP and CCE beads were collected and thoroughly washed with distilled water. 2.3. Antifungal activity assay Antifungal assays were performed as described previously [11] with slight modifications. Briefly, 17e20 chitosan beads at each concentration were placed on Petri dishes (Figs. 1A and 2A), and mycelial growth was measured after 1, 2, and 3 days of incubation. The inhibition percentage was calculated using the following formula:
Db Da 100 Relative activityð%Þ ¼ 1 Db where, Da is the diameter of the growth zone in the experimental dish (cm) and Db is the diameter of the growth zone in the control dish (cm).
Fig. 2. Antifungal activity of chitosanecinnamon extract (CCE) beads at different concentrations of cinnamon extract (CE) [T0: control, only chitosan beads (CTB), T1: CTB þ 0.1% CE, T2: CTB þ 0.3% CE, and T3: CTB þ 0.5% CE] against Rhizoctonia solani after 1, 2, and 3 days of incubation. (A) Mycelial growth of R. solani on potato dextrose agar (PDA) with CCE beads. (B) Relative activity (%) of the CCE beads. Different letters on the error bars indicate a significant difference based on Tukey’s Studentized range at p 0.05.
2.4. Nematicidal activity assay A direct-contact bioassay was used to evaluate M. incognita egg hatch after treatment with the chitosanecinnamon beads. Approximately 250 eggs in 1 mL of water and 10 beads of each concentration were added to each well of a 24-well Microtest tissue culture plate. The plate was covered with the original solid lid and wrapped with Parafilm, and the samples were kept at room temperature (25 1 C). Hatch inhibition was based on the number of hatched juveniles at 3 days after incubation, as observed under a light microscope (40). Approximately 250 J2 juveniles in 1 mL of water and 10 beads of each concentration were added to each well of a 24-well Microtest tissue culture plate to determine juvenile mortality caused by the chitosanecinnamon beads, and the samples were maintained at room temperature (25 1 C). At 0, 3, 6, 12, 24, and 48 h of incubation, dead and live nematodes were counted to evaluate mortality rates using a light microscope (40). J2 mortality was estimated according to the mean percentage of dead J2. Nematodes were considered dead when no movement was observed during 2 s after mechanical prodding [12]. Treatments were replicated six times, and the experiment was repeated. 3. Results and discussion
Fig. 1. Antifungal activity of chitosanecinnamon powder (CCP) beads with different concentrations of cinnamon powder (CP) [T0: control, only chitosan beads (CTB), T1: CTB þ 1% CP, T2: CTB þ 3% CP, and T3: CTB þ 5% CP] against Rhizoctonia solani after 1 and 2 days of incubation. (A) R. solani mycelial growth on potato dextrose agar (PDA) with CCP beads. (B) Relative activity (%) of the CCP beads. Different letters on the error bars indicate a significant difference based on Tukey’s Studentized range at p 0.05.
According to Nguyen et al. [7], fungal hyphae are completely destroyed when CE is applied directly to R. solani. Specifically, the cell walls deformed and collapsed, which resulted in the death of the hyphae in response to treatment with a 1% CE paste. Such
Please cite this article in press as: Seo D-J, et al., Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2013.12.007
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inhibitors are believed to block the synthesis of chitin in fungal cell walls [13]. Furthermore, cinnamon oil compounds exert activity against Dermatophagoides farinae and D. pteronyssinus when applied as fumigants [14], as well as antifungal activity against Oidium murrayae [15]. In this experiment, the inhibition of mycelial growth in response to chitosan-CCP beads showed in Fig. 1A. After a 1 and 2 d incubation, the relative activity of the CCP beads was 100, 72.4, 57.6, and 30.9% and that of CP was 100, 62.0, 47.8 and 23.9% at 0, 1, 3, and 5%, respectively (Fig. 1B). The relative activity of the CCE beads was significantly different among treatments with different concentrations of CE (Fig. 2). In particular, the inhibitory activity of the CCE beads still strongly affected R. solani mycelia growth after a 3-day incubation. The relative activity of the CCE beads was 100, 56.9, 9.0, and 4.2% at CE concentrations of 0, 0.1, 0.3, and 0.5%, respectively (Fig. 2B). In contrast, Nguyen et al. [7] demonstrated that the inhibitory activity of CE extracted with different organic solvents decreased gradually at 30 h compared to that at 10 h of incubation. These results suggest that chitosan beads combined with CP/CE could be used as a fungicide to control R. solani. In the present experiment, inhibition of M. incognita hatch by chitosanecinnamon (CCP and CCE) beads was evaluated using a direct-contact bioassay, and significant differences were observed with different cinnamon concentrations (Figs. 3A and 4A). The inhibition of hatch caused by the CCP beads was 26.0, 39.6, 63.3, and 78.8% at CP concentrations of 0, 1, 3, and 5%, respectively, after a 3-
Fig. 4. Nematicidal activity of chitosanecinnamon extract (CCE) beads against M. incognita with different concentrations of cinnamon extract (CE) [only chitosan beads (CTB), CTB þ 0.1% CE, CTB þ 0.3% CE, and CTB þ 0.5% CE]. (A) Inhibition of M. incognita hatch caused by the CCE beads after a 3-day incubation. (B) M. incognita juvenile (J2) mortality caused by the CCE beads after 0, 3, 6, 12, 24, and 48 h incubations. Different letters on the error bars indicate a significant difference based on Tukey’s Studentized range at p 0.05.
Fig. 3. Nematicidal activity of chitosanecinnamon powder (CCP) beads with different concentrations of cinnamon powder (CP) [only chitosan beads (CTB), CTB þ 1% CP, CTB þ 3% CP, and CTB þ 5% CP] against Meloidogyne incognita. (A) Inhibition of M. incognita hatch caused by the CCP beads after a 3-day incubation. (B) M. incognita juvenile (J2) mortality caused by the CCP beads after 0, 3, 6, 12, 24, and 48 h incubations. Different letters on the error bars indicate a significant difference based on Tukey’s Studentized range at p 0.05.
day incubation (Fig. 3A). The inhibition of hatch caused by the CCE beads was 21.0, 57.0, 79.0, and 82.0% at CE concentrations of 0, 0.1, 0.3, and 0.5%, respectively, after a 3-day incubation (Fig. 4A). Inhibition of hatch increased with increasing concentration, indicating dose-dependency. These results are significant because the egg stage is the most resistant stage in the nematode life cycle, possibly due to its three-layer shell [16,17]. Nematode eggs are surrounded by an eggshell, and strength is provided by the chitinous layer [18]. Abbas et al. [19] suggested that an aqueous extract of Cinnamomum tamala showed 100% (w/v) maximum nematicidal activity against Meloidogyne javanica eggs. An ethanol extract of C. tamala completely inhibited hatching of M. javanica eggs at 1000 ppm after a 3 days exposure. Additionally, treatment with a 1000 ppm ethanol extract of C. tamala resulted in maximum killing of juveniles. Salgado and Campos [20] showed that Cinnamomum zeylanicum solutions cause 100% mortality of Meloidogyne exigua after a 24 h treatment. In the present study, bioassays of chitosanecinnamon (CCP and CCE) beads revealed significant differences in toxicity against M. incognita juveniles (Figs. 3 and 4). J2 mortality caused by the chitosanecinnamon beads was 7.5, 52.5, 65.5, and 85.0% at CP concentrations of 0, 1, 3, and 5%, respectively, after a 48 h incubation (Fig. 3B) and was 6.7, 53.3, 83.3 and 95.8% at CE concentrations of 0, 0.1, 0.3, and 0.5%, respectively after a 48 h incubation (Fig. 4B). Nguyen et al. [9] reported that J2 mortality was
Please cite this article in press as: Seo D-J, et al., Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2013.12.007
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100% after only a 50-min incubation with 0.1 mg/mL cinnamyl acetate purified from cinnamon. Overall, J2 mortality increased with increasing concentration and time. These results suggest that chitosanecinnamon beads may be considered a drug carrier, because the nematicidal activity against M. incognita was prolonged. In conclusion, a chitosanecinnamon bead conjugate with nematicidal and antifungal activity was bioassayed. We found that the chitosanecinnamon beads could be considered a bio-pesticides carrier. However, further studies are needed to evaluate the fumigant effects under field conditions for practical use of the chitosane cinnamon beads as novel fumigants or hatching inhibitors. If commercial bio-pesticides are used as biological control agents for soil-borne phytopathogens, formulations and stability that improve their nematicidal and antifungal activities must be developed to reduce costs and make them practical for use in the field. Acknowledgements This study was financially supported by Chonnam National University, 2012. References [1] Juang RS, Wu FC, Tseng RL. Solute adsorption and enzyme immobilization on chitosan beads prepared from shrimp shell wastes. Bioresour Technol 2011;80:187e93. [2] Zubriene A, Budriene S, Lubiene J, Dienys G. Immobilized alkaline phosphatase for molecular cloning. Biocatal Biotransform 2002;20:423e7. [3] Yu SH, Mi FL, Pang JC, Jiang SC, Kuo TH, Wu SJ, et al. Preparation and characterization of radical and pH-responsive chitosan-gallic acid conjugate drug carriers. Carbohyd Polym 2011;84:794e802. [4] WHO/WPRO. Medicinal plants in Vietnam. Hanoi: Institute of Materia Medica; 1990. p. 444. [5] Nielsen PV, Rios R. Inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application in active packaging, with special emphasis on mustard essential oil. Int J Food Microbiol 2000;60:219e29.
[6] Nguyen DMC, Nguyen VN, Seo DJ, Park RD, Jung WJ. Nematicidal activity of compounds extracted from medicinal plants against the pine wood nematode Bursaphelenchus xylophilus. Nematology 2009a;11:835e45. [7] Nguyen VN, Nguyen DMC, Seo DJ, Park RD, Jung WJ. Antimycotic activities of cinnamon-derived compounds against Rhizoctonia solani in vitro. BioControl 2009b;54:697e707. [8] Nguyen DMC, Seo DJ, Park RD, Lee BR, Jung WJ. Changes in antioxidative enzymes in cucumber plants with regard to biological control of root-knot nematode, Meloidogyne incognita with Cinnamomum cassia crude extracts. J Korean Soc Appl Biol Chem 2011;54:507e14. [9] Nguyen DMC, Seo DJ, Kim KY, Kim TH, Jung WJ. Nematode-antagonistic effects of Cinnamomum aromaticum extracts and a purified compound against Meloidogyne incognita. Nematology 2012;14:913e24. [10] Mitani T, Fukumuro N, Yoshimoto C, Ishii H. Effects of ions (SO2 4 and Cl ) on the adsorption of copper and nickel ions by swollen chitosan beads. Agric Biol Chem 1991;55:2419. [11] Soylu EM, Soylu S, Kurt S. Antimicrobial activities of the essential oils of various plants against tomato late blight disease agent Phytophthora infestans. Mycopathologia 2006;161:119e28. [12] Cayrol JC, Djian C, Pijarowski L. Study on the nematicidal properties of the culture filtrate of the nematophagus fungus Paecilomyces lilacinus. Rev Nematol 1989;12:331e6. [13] Liu YJ, Zhang KQ. Antimicrobial activity of selected Cyathus species. Mycopathologia 2004;157:185e9. [14] Kim HK, Yun YK, Ahn YJ. Fumigant toxicity of cassia bark and cassia and cinnamon oil compounds to Dermatophagoides farinae and Dermatophagoides pteronyssinus (Acari: Pyroglyphidae). Exp Appl Acarol 2008;44:1e9. [15] Chu YL, Ho WC, Ko WH. Effects of Chinese herb extracts on spore germination of Oidium murrayae and nature of inhibition substances from Chinese Rhubarb. Plant Dis 2006;90:848e61. [16] Wharton DA. Nematode survival strategies. In: Lee DL, editor. The biology of nematodes. New York, NY, USA: Taylor and Francis; 2002. pp. 389e411. [17] Harris MT, Fuhrman JA. Structure and expression of chitin synthase in the parasitic nematode Dirofilaria immitis. Mol Biochem Parasit 2002;122:231e4. [18] Fanelli E, Di Vito M, Jones JT, De Giorgi C. Analysis of chitin synthase function in a plant parasitic nematode, Meloidogyne artiellia, using RNAi. Gene 2005;349:87e95. [19] Abbas S, Dawar S, Tariq M, Zaki MJ. Nematicidal activity of spices against Meloidogyne javanica (Treub) Chitwood. Pakistan J Bot 2009;41: 2626e32. [20] Salgado SML, Campos VP. Hatching and mortality of Meloidogyne exigua in extracts and in natural products. Fitopatol Brasil 2003;28:166e70.
Please cite this article in press as: Seo D-J, et al., Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2013.12.007
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Short communication
Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro Q1
Dong-Jun Seo a, Dang-Minh-Chanh Nguyen a, b, Ro-Dong Park a, Woo-Jin Jung a, * a Division of Applied Bioscience and Biotechnology, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Science, Chonnam National University, Gwangju 500-757, Republic of Korea b Western Highlands Agriculture Forestry Science Institute, 53 Nguyen Luong Bang Street, Buon Ma Thuot, Vietnam
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 October 2013 Received in revised form 25 December 2013 Accepted 30 December 2013 Available online xxx
A novel chitosanecinnamon bead carrier was prepared in this study. Chitosan was mixed with cinnamon powder (CP) and cinnamon extract (CE) to obtain chitosanecinnamon powder (CCP) beads and chitosan ecinnamon extracted (CCE) beads, respectively. The potential antifungal and nematicidal activities of CCP and CCE were investigated against Rhizoctonia solani and Meloidogyne incognita in vitro. Relative antifungal activity of the CCP (5% CP) bead-treated R. solani was 30.9 and 23.9% after 1 and 2 day incubations, respectively. Relative antifungal activity of the CCE (0.5% CE) bead-treated R. solani was 4.3, 3.0 and 4.2% after 1, 2 and 3 days of incubation. Inhibition of hatch by CCP beads with CP of 5% was 78.8%. Inhibition of hatch by CCE beads with CE of 0.5% was 82.0%. J2 mortality following the CCP (5% CP) and CCE (0.5% CE) bead treatments was 85.0 and 95.8%, respectively against M. incognita after 48 h incubations. Ó 2014 Published by Elsevier Ltd.
Keywords: Chitosan beads Cinnamomum cassia Antifungal activity Nematicidal activity
1. Introduction Chitosan is comprised of linear high molecular weight polyglucosamine chains and possesses distinct chemical and biological properties. Therefore, chitosan has been used as a swollen bead support for preparing immobilized enzymes and conjugate drug carriers [1e3]. Cinnamon (Cinnamomum cassia) is a tall (10e20 m) native medicinal plant that grows in the Central Highlands of Vietnam. The tree bark is harvested throughout the year, preferably in summer and autumn. It is applied as therapy for dyspepsia, colic, diarrhoea, cholera, dysentery, coryza, influenza, cough, asthma, paresis, and snake-bites [4]. Additionally, cinnamon extracts are known for their antifungal activity and are used in bakery products for their aromatic properties. Nielsen and Rios [5] demonstrated that cinnamon extracts exert antifungal effects through their volatile components, and reported that they have a potential application in active packaging. Previous studies have suggested that crude cinnamon extracts exert nematicidal activity against the pine wood nematode, Bursaphelenchus xylophilus [6]. Thus, methanol extracts of cinnamon and their sub-fractions could be useful as fungicides against Rhizoctonia solani [7]. Additionally, Nguyen et al. [8] showed that antioxidant enzyme activity such as superoxide dismutase,
* Corresponding author. Tel.: þ82 62 530 3960, fax: þ82 62 530 2139. E-mail address: [email protected] (W.-J. Jung).
catalase, and ascorbate peroxidase increase in the leaves of cucumber plants treated with 5 or 10 mg/mL C. cassia extract compared with leaves of untreated plants on day 28 after treatment. These findings suggest that the induction of pathogenesisrelated proteins following C. cassia extract treatment may strengthen the plants against various biotic stressors. Furthermore, cinnamyl acetate purified from cinnamon shows high nematicidal activity against Meloidogyne incognita [9]. In this study, chitosan beads were used as carriers to immobilize the cinnamon activities. Applying cinnamon to swollen chitosan beads has not been extensively studied. This study was designed to investigate the activities of cinnamon combined with swollen chitosan beads against the phytopathogens R. solani and M. incognita in vitro. 2. Materials and methods 2.1. Materials Chitosan powder [90% deacetylation, 10 cPs (in 0.5% acetic acid þ 0.5% chitosan solution, at 20 C)] was purchased from Keumho Chemical Co., Ltd. (Seoul, Korea). C. cassia bark was purchased from Buon Ma Thuot market, a medicinal plant and herb shop in DakLak province, Vietnam. R. solani KACC 40111 was obtained from the Korea Agricultural Culture Collection (Suwon, Korea). M. incognita eggs and second-stage juveniles (J2) were
0882-4010/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.micpath.2013.12.007
Please cite this article in press as: Seo D-J, et al., Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2013.12.007
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extracted from infected cucumber roots in a growth chamber. All other chemicals were of analytical grade. 2.2. Preparation of chitosanecinnamon powder (CCP) and chitosanecinnamon extract (CCE) beads The cinnamon bark was cut into 0.5e1.0 cm pieces and powdered using a mortar and pestle to obtain the cinnamon powder (CP). The cinnamon bark was cut into 2e3 cm pieces and extracted with 99% methanol at a ratio of 1:5 (v/v, dry bark/methanol) at 30 C with shaking at 150 rpm for 7 days. The filtrate was evaporated to dryness under a vacuum at 40 C (Eyela N-1000, Tokyo, Japan) and then lyophilized to obtain the cinnamon extract (CE). The CCP was prepared by dissolving chitosan powder (7 g) in 100 mL of 7% acetic acid and then adding it to 0, 1, 3, and 5 g CP, respectively. Similarly, the CCE was prepared by dissolving chitosan powder (7 g) in 100 mL of 7% acetic acid and then 0, 0.1, 0.3, and 0.5 g of CE were added. The resulting viscous solution of CCP and CCE was de-gassed under vacuum and dropped into 200 mL of an alkali coagulating solution (H2OeMeOHeNaOH ¼ 4:5:1, w/w/w) to prepare highly swollen 3.5 mm average diameter spherical beads [10]. The CCP and CCE beads were collected and thoroughly washed with distilled water. 2.3. Antifungal activity assay Antifungal assays were performed as described previously [11] with slight modifications. Briefly, 17e20 chitosan beads at each concentration were placed on Petri dishes (Figs. 1A and 2A), and mycelial growth was measured after 1, 2, and 3 days of incubation. The inhibition percentage was calculated using the following formula:
Db Da 100 Relative activityð%Þ ¼ 1 Db where, Da is the diameter of the growth zone in the experimental dish (cm) and Db is the diameter of the growth zone in the control dish (cm).
Fig. 2. Antifungal activity of chitosanecinnamon extract (CCE) beads at different concentrations of cinnamon extract (CE) [T0: control, only chitosan beads (CTB), T1: CTB þ 0.1% CE, T2: CTB þ 0.3% CE, and T3: CTB þ 0.5% CE] against Rhizoctonia solani after 1, 2, and 3 days of incubation. (A) Mycelial growth of R. solani on potato dextrose agar (PDA) with CCE beads. (B) Relative activity (%) of the CCE beads. Different letters on the error bars indicate a significant difference based on Tukey’s Studentized range at p 0.05.
2.4. Nematicidal activity assay A direct-contact bioassay was used to evaluate M. incognita egg hatch after treatment with the chitosanecinnamon beads. Approximately 250 eggs in 1 mL of water and 10 beads of each concentration were added to each well of a 24-well Microtest tissue culture plate. The plate was covered with the original solid lid and wrapped with Parafilm, and the samples were kept at room temperature (25 1 C). Hatch inhibition was based on the number of hatched juveniles at 3 days after incubation, as observed under a light microscope (40). Approximately 250 J2 juveniles in 1 mL of water and 10 beads of each concentration were added to each well of a 24-well Microtest tissue culture plate to determine juvenile mortality caused by the chitosanecinnamon beads, and the samples were maintained at room temperature (25 1 C). At 0, 3, 6, 12, 24, and 48 h of incubation, dead and live nematodes were counted to evaluate mortality rates using a light microscope (40). J2 mortality was estimated according to the mean percentage of dead J2. Nematodes were considered dead when no movement was observed during 2 s after mechanical prodding [12]. Treatments were replicated six times, and the experiment was repeated. 3. Results and discussion
Fig. 1. Antifungal activity of chitosanecinnamon powder (CCP) beads with different concentrations of cinnamon powder (CP) [T0: control, only chitosan beads (CTB), T1: CTB þ 1% CP, T2: CTB þ 3% CP, and T3: CTB þ 5% CP] against Rhizoctonia solani after 1 and 2 days of incubation. (A) R. solani mycelial growth on potato dextrose agar (PDA) with CCP beads. (B) Relative activity (%) of the CCP beads. Different letters on the error bars indicate a significant difference based on Tukey’s Studentized range at p 0.05.
According to Nguyen et al. [7], fungal hyphae are completely destroyed when CE is applied directly to R. solani. Specifically, the cell walls deformed and collapsed, which resulted in the death of the hyphae in response to treatment with a 1% CE paste. Such
Please cite this article in press as: Seo D-J, et al., Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2013.12.007
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inhibitors are believed to block the synthesis of chitin in fungal cell walls [13]. Furthermore, cinnamon oil compounds exert activity against Dermatophagoides farinae and D. pteronyssinus when applied as fumigants [14], as well as antifungal activity against Oidium murrayae [15]. In this experiment, the inhibition of mycelial growth in response to chitosan-CCP beads showed in Fig. 1A. After a 1 and 2 d incubation, the relative activity of the CCP beads was 100, 72.4, 57.6, and 30.9% and that of CP was 100, 62.0, 47.8 and 23.9% at 0, 1, 3, and 5%, respectively (Fig. 1B). The relative activity of the CCE beads was significantly different among treatments with different concentrations of CE (Fig. 2). In particular, the inhibitory activity of the CCE beads still strongly affected R. solani mycelia growth after a 3-day incubation. The relative activity of the CCE beads was 100, 56.9, 9.0, and 4.2% at CE concentrations of 0, 0.1, 0.3, and 0.5%, respectively (Fig. 2B). In contrast, Nguyen et al. [7] demonstrated that the inhibitory activity of CE extracted with different organic solvents decreased gradually at 30 h compared to that at 10 h of incubation. These results suggest that chitosan beads combined with CP/CE could be used as a fungicide to control R. solani. In the present experiment, inhibition of M. incognita hatch by chitosanecinnamon (CCP and CCE) beads was evaluated using a direct-contact bioassay, and significant differences were observed with different cinnamon concentrations (Figs. 3A and 4A). The inhibition of hatch caused by the CCP beads was 26.0, 39.6, 63.3, and 78.8% at CP concentrations of 0, 1, 3, and 5%, respectively, after a 3-
Fig. 4. Nematicidal activity of chitosanecinnamon extract (CCE) beads against M. incognita with different concentrations of cinnamon extract (CE) [only chitosan beads (CTB), CTB þ 0.1% CE, CTB þ 0.3% CE, and CTB þ 0.5% CE]. (A) Inhibition of M. incognita hatch caused by the CCE beads after a 3-day incubation. (B) M. incognita juvenile (J2) mortality caused by the CCE beads after 0, 3, 6, 12, 24, and 48 h incubations. Different letters on the error bars indicate a significant difference based on Tukey’s Studentized range at p 0.05.
Fig. 3. Nematicidal activity of chitosanecinnamon powder (CCP) beads with different concentrations of cinnamon powder (CP) [only chitosan beads (CTB), CTB þ 1% CP, CTB þ 3% CP, and CTB þ 5% CP] against Meloidogyne incognita. (A) Inhibition of M. incognita hatch caused by the CCP beads after a 3-day incubation. (B) M. incognita juvenile (J2) mortality caused by the CCP beads after 0, 3, 6, 12, 24, and 48 h incubations. Different letters on the error bars indicate a significant difference based on Tukey’s Studentized range at p 0.05.
day incubation (Fig. 3A). The inhibition of hatch caused by the CCE beads was 21.0, 57.0, 79.0, and 82.0% at CE concentrations of 0, 0.1, 0.3, and 0.5%, respectively, after a 3-day incubation (Fig. 4A). Inhibition of hatch increased with increasing concentration, indicating dose-dependency. These results are significant because the egg stage is the most resistant stage in the nematode life cycle, possibly due to its three-layer shell [16,17]. Nematode eggs are surrounded by an eggshell, and strength is provided by the chitinous layer [18]. Abbas et al. [19] suggested that an aqueous extract of Cinnamomum tamala showed 100% (w/v) maximum nematicidal activity against Meloidogyne javanica eggs. An ethanol extract of C. tamala completely inhibited hatching of M. javanica eggs at 1000 ppm after a 3 days exposure. Additionally, treatment with a 1000 ppm ethanol extract of C. tamala resulted in maximum killing of juveniles. Salgado and Campos [20] showed that Cinnamomum zeylanicum solutions cause 100% mortality of Meloidogyne exigua after a 24 h treatment. In the present study, bioassays of chitosanecinnamon (CCP and CCE) beads revealed significant differences in toxicity against M. incognita juveniles (Figs. 3 and 4). J2 mortality caused by the chitosanecinnamon beads was 7.5, 52.5, 65.5, and 85.0% at CP concentrations of 0, 1, 3, and 5%, respectively, after a 48 h incubation (Fig. 3B) and was 6.7, 53.3, 83.3 and 95.8% at CE concentrations of 0, 0.1, 0.3, and 0.5%, respectively after a 48 h incubation (Fig. 4B). Nguyen et al. [9] reported that J2 mortality was
Please cite this article in press as: Seo D-J, et al., Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2013.12.007
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100% after only a 50-min incubation with 0.1 mg/mL cinnamyl acetate purified from cinnamon. Overall, J2 mortality increased with increasing concentration and time. These results suggest that chitosanecinnamon beads may be considered a drug carrier, because the nematicidal activity against M. incognita was prolonged. In conclusion, a chitosanecinnamon bead conjugate with nematicidal and antifungal activity was bioassayed. We found that the chitosanecinnamon beads could be considered a bio-pesticides carrier. However, further studies are needed to evaluate the fumigant effects under field conditions for practical use of the chitosane cinnamon beads as novel fumigants or hatching inhibitors. If commercial bio-pesticides are used as biological control agents for soil-borne phytopathogens, formulations and stability that improve their nematicidal and antifungal activities must be developed to reduce costs and make them practical for use in the field. Acknowledgements This study was financially supported by Chonnam National University, 2012. References [1] Juang RS, Wu FC, Tseng RL. Solute adsorption and enzyme immobilization on chitosan beads prepared from shrimp shell wastes. Bioresour Technol 2011;80:187e93. [2] Zubriene A, Budriene S, Lubiene J, Dienys G. Immobilized alkaline phosphatase for molecular cloning. Biocatal Biotransform 2002;20:423e7. [3] Yu SH, Mi FL, Pang JC, Jiang SC, Kuo TH, Wu SJ, et al. Preparation and characterization of radical and pH-responsive chitosan-gallic acid conjugate drug carriers. Carbohyd Polym 2011;84:794e802. [4] WHO/WPRO. Medicinal plants in Vietnam. Hanoi: Institute of Materia Medica; 1990. p. 444. [5] Nielsen PV, Rios R. Inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application in active packaging, with special emphasis on mustard essential oil. Int J Food Microbiol 2000;60:219e29.
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Please cite this article in press as: Seo D-J, et al., Chitosanecinnamon beads enhance suppressive activity against Rhizoctonia solani and Meloidogyne incognita in vitro, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2013.12.007
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