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Chitosan beads combined with Terminalia nigrovenulosa bark enhance suppressive activity to Fusarium solani
Industrial Crops and Products 50 (2013) 462–467
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Chitosan beads combined with Terminalia nigrovenulosa bark enhance suppressive activity to Fusarium solani Dang-Minh-Chanh Nguyen a,b , Dong-Jun Seo a , Ro-Dong Park a , Byung-Jin Lee c , 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, Viet Nam c Department of Agronomy, Gyeongnam National University of Science and Technology, Chilam-Dong 150, Jinju-City, Republic of Korea
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Article history: Received 27 March 2013 Received in revised form 2 July 2013 Accepted 25 July 2013 Keywords: Terminalia nigrovenulosa Chitosan beads Biocontrol Fusarium solani Pathogenesis-related proteins
a b s t r a c t Chitosan beads (CTB) and Terminalia nigrovenulosa bark (TNB) were prepared using chitosan powder combined with T. nigrovenulosa powder (TNP) and T. nigrovenulosa extract (TNE) to obtain CTB + TNP (0, 1, 3, and 5%) and CTB + TNE (0, 0.1, 0.3, and 0.5%), respectively. The chitosan beads combined with TNB were tested for their antifungal activity against Fusarium solani. In vitro studies indicated that a mixture of 20 chitosan beads and 3–5% TNP inhibited F. solani mycelial growth the greatest. Mixtures of 10–20 chitosan beads and 0.3–0.5% TNE showed the greatest inhibition of F. solani mycelial growth. Total inhibition of F. solani mycelial growth was achieved with CTB and 5% TNP and CTB and 0.5% TNE after a 6 day incubation. Induction of pathogenesis-related proteins such as chitinase, -1,3-glucanase, and guaiacol peroxidase were tested in cucumber seedlings. Enzyme accumulation was significantly higher in CTB and 0.5% TNE treated cucumber plants compared to that in control plants. We effectively demonstrated the biological activities of the CTB and TNP and CTB and TNE treatments. These results suggest that applying chitosan beads combined with TNB could be useful to control soil-borne diseases of cucumber seedlings. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Diseases caused by Fusarium solani are a limiting factor in plant production, and yield quantity. F. solani causes the death of young and adult plants, with consequent economic losses (Kim and Jee, 1998). Several effective fungicides have been recommended for use against pathogens, but they are not considered long-term solutions due to expense, exposure risk, fungicide residue, and environmental hazard concerns. Therefore, alternative control methods that can be safely and effectively used are urgently required (Kavino et al., 2008). Applying plant extracts and biocontrol agents is eco-friendly and effective against many plant pathogens (Kagale et al., 2004). A number of plant species possess natural substances that are toxic to many pests and pathogens (Akhtar et al., 2008; Harish et al., 2008). These pesticides are generally considered non-persistent under field conditions, as they are readily transformed by light, oxygen, or microorganisms into less toxic products. Therefore, fewer residues are expected from those products (Akhtar et al., 2008). Up to date, various plant extracts exert different levels of antifungal activity in vitro against phytopathogenic fungi (Soylu et al.,
∗ Corresponding author. Tel.: +82 62 530 3960; fax: +82 62 530 2139. E-mail address: [email protected] (W.-J. Jung). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.07.060
2006; Jasso de Rodríguez et al., 2005; Bajwa and Iftikhar, 2005). Chitosan is obtained by partial deacetylation of chitin (Muzzarelli, 2009; Jayakumar et al., 2010). Chitosan has been used as a swollen bead support for preparing immobilized enzymes and conjugate drug carriers (Juang et al., 2001; Yu et al., 2011). Chitosan as fungicides are effective in inhibiting spore germination, germ tube elongation and mycelial growth of fungal phytopathogens, such as Phytophthora capsici and Alternaria solani (Xu et al., 2007), Fusarium (Eweis et al., 2006), and Alternaria kikuchiana and Physalospora piricola (Meng et al., 2010). Additionally, combinations of chitosan and Cornus officinalis seed extract have been showed high antifungal activities, especially against Rhizoctonia solani and F. solani (Seo et al., 2013). Plants in the genus Terminalia, family Combretaceae, comprising some 250 species, are widely distributed in tropical areas of the world (Fabry et al., 1998). A methanol extract of Terminalia arjuna and T. superba bark shows nematicidal and antifungal activities against Haemonchus contortus, Trychophyton rubrum, and Microsporum audouinii (Bachaya et al., 2009; Kuete et al., 2010). Additionally, the presence of phenolic acids such as gallic acid, ellagic acid, and corilagin acid is one of the reasons for the antifungal nature of the Terminalia chebula Retz (Rangsriwong et al., 2009). In our previous studies, Terminalia nigrovenulosa bark (TNB) showed strong biocontrol properties against F. solani (Nguyen et al.,
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2013b) and Meloidogyne incognita (Nguyen et al., 2013a) in vitro. In this study, chitosan beads were used as a carrier to immobilize the agent showing both antifungal and nematicidal activities of TNB. Applying TNB to swollen chitosan beads has not been studied extensively. This study was designed to investigate the activities of TNB combined with swollen chitosan beads against the phytopathogens F. solani. 2. Materials and methods 2.1. Materials Chitosan powder [90% deacetylation, 10 cPs (in 0.5% acetic acid + 0.5% chitosan solution, at 20 ◦ C)] was purchased from Keumho Chemical Co., Ltd. (Seoul, Korea). TNB was collected from DakLak province, Vietnam. F. solani KACC 40384 was obtained from the Korea Agricultural Culture Collection (Suwon, Korea). All other chemicals were of analytical grade. 2.2. Preparation of chitosan beads (CTB) combined with T. nigrovenulosa powder (TNP) and CTB combined with T. nigrovenulosa extract (TNE)
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randomized design under chamber conditions. All procedures were performed under sterile conditions. 2.5. Activity measurement of pathogenesis-related (PR) proteins At 7 days after pathogen inoculation, leaves sample was homogenized with liquid nitrogen in a pre-chilled mortar and pestle. 200 mg of sample was homogenized with 1 mL of 50 mM Tris buffer (pH 6.7), and the homogenate was centrifuged at 10,000 × g for 20 min at 4 ◦ C. The supernatant was used as a crude enzyme extract for assaying chitinase, -1,3-glucanase and guaiacol-peroxidase (GPOD). Protein concentration was determined according to the method described by Bradford (1976) using bovine serum albumin as a standard. Chitinase activity was assayed by measuring the reducing end group, GlcNAc (N-acetyl--d-glucosamine), produced by colloidal chitin (Lingappa and Lockwood, 1962). 1,3-glucanase activity was assayed by measuring the amount of the reducing end group, glucose, produced from laminarin (Yedidia et al., 2000). GPOD activity was assayed by measuring the amount of the reducing end group produced using the method described by Jinmin and Bingru (2001). 2.6. Activity staining of PR proteins
TNB was cut into 0.5–1.0 cm pieces and powdered using a mortar and pestle to obtain TNP. The TNB was also cut into 2–3 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) and then lyophilized to obtain the TNE. The mixtures of CTB and TNP were 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 TNP, respectively. Similarly, mixtures of CTB and TNE were 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 TNE was added, respectively. The resulting viscous solutions of CTB and TNP and CTB and TNE were de-gassed under vacuum and dropped into 200 mL of an alkali coagulating solution (H2 O–MeOH–NaOH = 4:5:1, w/w/w) to prepare highly swollen 3.5 mm average diameter spherical beads (Mitani et al., 1991). The chitosan beads were collected and thoroughly washed with distilled water. 2.3. Testing the antifungal spectrum of TNB using Petri dish Antifungal assays were performed as previously described by Soylu et al. (2006) with slight modifications. The antifungal spectrum was determined by placing a 4 mm-diameter agar disk cut from the edge of an actively growing colony of the test pathogen onto the center of a new Petri dish containing potato dextrose agar medium. Subsequently, the 5, 10, and 20 CTBs of each concentration were placed carefully around a F. solani colony on a PDA plate, respectively. Each combination was tested using three replicates. Zones of inhibition were measured at 2, 4, and 6 days after incubation in the dark at 25 ◦ C. The test was conducted twice. 2.4. Antifungal activity of TNB for disease control Cucumber (C. sativus L. Asia Unchun F1) seeds were sown in steam sterilized soil in 5 cm clay pots, and single seedlings were transplanted into each of the pots containing 200 g of sterile soil 1 week after germination. All seedlings were kept in a growth chamber under a 16/8 h light/dark photoperiod, a 26/20 ◦ C day/night and 60–70% relative humidity. The plants were inoculated with spore suspensions (2 × 105 spores/mL) of F. solani. Subsequently, the cucumber plants were treated with 20 beads of chitosan and TNE (0, 0.1, 0.3, and 0.5%). Three replications were maintained for each treatment, and each replication consisted of three pots in a
Thirty micrograms of enzyme extracts was loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE gels to detect -1,3-glucanase and GPOD activity, respectively. For active staining of -1,3-glucanase after 12% (w/v) SDS-PAGE, a gel was prepared by dipping it in 0.15% triphenyltetrazolium chloride solution containing 1 N NaOH followed by heating in an oven until red bands appeared (Pan et al., 1989). The gels were soaked in 50 mM Tris buffer (pH 6.8) for 10 min to actively stain GPOD after 10% (w/v) native PAGE, and then they were incubated with 0.46% (v/v) guaiacol and 13 mM H2 O2 in the same buffer until red bands appeared. The gels were fixed in water/methanol/acetic acid (6.5:2.5:1, v/v) (Caruso et al., 1999). 2.7. Statistical analysis Data on the effect of the treatments on the growth of pathogens and activity of enzymes in cucumber plants were compared using Tukey’s Studentized range (HSD) test, with a p ≤ 0.05 indicating statistical significance. All data were analyzed using the Statistical Analysis System 9.1 (SAS Institute, Cary, NC, USA) and are presented as mean ± standard deviation. 3. Results and discussion In this study, mixtures of chitosan beads and TNB inhibited growth of F. solani. F. solani mycelia grew slowly on PDA plates containing CTB and TNP (Fig. 1a–c). In particular, the mycelial disk of F. solani treated with 20 CTB and 3% or 5% TNP did not grow for 2, 4, or 6 days (Fig. 1c, T2 and T3). Inhibition of 5 CTB with all tested concentrations against F. solani was only moderate (Fig. 1a). Briefly, mycelial growth was 46.3, 28.7, 18.7, and 18.3 mm at 0, 1, 3, and 5% of TNP, respectively for a 6 day treatment with the 5 CTB (Fig. 1d). Mycelial growth was 46.3, 15.0, 7.7, and 7.7 mm at CTB and 0, 1, 3, and 5% TNP with 10 CTB, respectively after 6 days of treatment (Fig. 1e). The 20 CTB managed to inhibit the mycelial growth of F. solani completely (Fig. 1f). Similary, the results of tests with CTB and TNE revealed that the inhibition of the 5 CTB and TNE (0, 0.1, 0.3, and 0.5%) against F. solani was only moderate (Fig. 2a and d). The 10 CTB and 0.3 or 0.5% TNE significantly inhibited mycelial growth after 4 days of treatment (Fig. 2b and e). The 20 CTB and 0.3 or 0.5% TNE inhibited mycelial growth completely after 6 days of treatment (Fig. 2c and f).
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Fig. 1. Antifungal activity of chitosan beads (CTB) combined with Terminalia nigrovenulosa powder (TNP) against F. solani at 2, 4, and 6 day after incubation. T0: CTB + 0% TNP, T1: CTB + 1% TNP, T2: CTB + 3% TNP, and T3: CTB + 5% TNP. Mycelial growth of F. solani on potato dextrose agar (PDA) treated with five (a and d), ten (b and e), and 20 chitosan beads (c and f). Different letters above the standard error bars indicate a significant difference at each observation time based on Tukey’s Studentized range at p ≤ 0.05.
A concentration of 10% zimmu leaf extract is highly effective for reducing mycelial growth (by 87%) of Alternaria solani (Latha et al., 2009). Kuete et al. (2010) suggested that a methanol extract of Terminalia superba has antifungal activities against Trychophyton rubrum and Microsporum audouinii. Bhutta et al. (2001) tested 32 different seeds and found that seeds from Coriander sativum and Memoranda charata revealed inhibitory effects at 0.5–1.0% concentrations against Alternaria alternata and F. solani. The presence of phenolic acids such as gallic acid, ellagic acid, and corilagin acid are one of the reasons responsible for the antifungal nature of T. chebula Retz (Rangsriwong et al., 2009). Bianchi et al. (1997) reported that the use of garlic micronized powder inhibited mycelial growth of F. solani and R. solani. Eksteen et al. (2001) tested 11 plant extracts against F. oxysporum and R. solani by the agar
dilution method and inhibited mycelial growth comparable to that obtained using carbendazim and difenconazole. In this study, the degree of inhibition varied significantly (p ≤ 0.05) according to the TNB concentrations. Thus, we suggest that in vitro antifungal activity of chitosan combined with TNB was dose-dependent because antifungal activity increased with increasing concentration of TNB. Fig. 3 shows that the mixtures of chitosan beads and TNE significantly inhibited the root system of cucumber plants caused by F. solani. Control plants were treated with CTB but without TNE, which caused complete stunting and root-rot infection (Fig. 3, T0, arrow). In contrast, plants treated with a mixture of CTB and TNE showed root system growth and development normally. In particular, plants treated with mixtures of chitosan beads and TNE (0.5%) showed well developed root systems and leaves (Fig. 3, T3). These
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Fig. 2. Antifungal activity of chitosan beads (CTB) combined with Terminalia nigrovenulosa extract (TNE) against F. solani at 2, 4, and 6 day after incubation. T0: CTB + 0% TNE, T1: CTB + 0.1% TNE, T2: CTB + 0.3% TNE, and T3: CTB + 0.5% TNE. Mycelial growth of F. solani on potato dextrose agar (PDA) treated with five (a and d), ten (b and e), and 20 chitosan beads (c and f). Different letters above the standard error bars indicate a significant difference at each observation time based on Tukey’s Studentized range at p ≤ 0.05.
results agree with those of Lee et al. (1995), who investigated the antigrowth and cytotoxic effects of gallic acid and chebulinic acid in a Terminalia chebula extract. Nguyen et al. (2013b) also showed that treatment with 1000 ppm of TNB resulted in 84% suppression of Fusarium root rot, while control plants were not treated with TNB but inoculated with F. solani alone, which induced up to 100% root rot, with the majority of plants completely stunted. Sundaramoorthy et al. (2012) provided evidence that Pseudomonas fluorescens and Bacillus subtilis effectively inhibit the growth of F. solani in chili plants. One of the mechanisms by which compounds exert their antifungal activity is through the degradation of pathogenic fungi cell walls. Such inhibitors are believed to block the synthesis of chitin
in fungal cell walls (Liu and Zhang, 2004). Chitinolytic enzymes are considered important in the biological control of soil-borne pathogens (Singh et al., 1999; Kim et al., 2008; Latha et al., 2009). PR proteins such as chitinase and -1,3-glucanase protect plants by degrading the walls of pathogenic fungal cells. Kim and Hwang (1994) reported that injecting pepper stems with a pathogenic strain, Phytophthora capsici, induced these PR proteins. Silva et al. (2004) reported that high peroxidase activities occur in pathogeninfected tomato plants during the later stages of infection. In this study, disease resistance in cucumber plants treated with chitosan beads and TNE revealed higher activity and expression of pathogenesis-related proteins against root-rot pathogen. Increased chitinase, -1,3-glucanase and GPOD activities were observed in
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Fig. 3. The efficacy of chitosan beads (CTB) combined with Terminalia nigrovenulosa extract (TNE) on the suppression of root rot diseases by Fusarium solani at 7 days after treatment. T0: F. solani + CTB (0% TNE); T1: F. solani + CTB (0.1% TNE); T2: F. solani + CTB (0.3% TNE); T3: F. solani + CTB (0.5% TNE).
Table 1 Pathogenesis-related protein activity of cucumber leaves in plants treated with Fusarium solani and chitosan beads with different concentrations of Terminalia nigrovenulosa extract (TNE) at 7 days after treatment. Treatment
PR-proteins activity Chitinase (Unit/g Fw)
T0 T1 T2 T3
1.03 1.55 3.38 4.45
± ± ± ±
0.40c 0.50bc 0.73ab 1.06a
-1,3-glucanase (Unit/g Fw) 17.00 23.56 29.04 50.83
± ± ± ±
5.60b 4.07b 5.94b 5.53a
GPOD (Unit/g Fw) 2.56 3.58 4.41 4.78
± ± ± ±
0.60b 0.77ab 0.43a 0.37a
Values shown in each column are means ± standard deviations based on three replicates. The same letters on each column indicate no significant difference based on Tukey’s Studentized range at p ≤ 0.05. T0: F. solani + CTB (0% TNE); T1: F. solani + CTB (0.1% TNE); T2: F. solani + CTB (0.3% TNE); T3: F. solani + CTB (0.5% TNE).
the CTB and TNE treated plants inoculated with F. solani compared to those in untreated control plants at 7 days after inoculation (Table 1). Chitinase activity of plants that were treated with CTB and 0.5% TNB increased significantly to 4.45 Unit/g Fw, compared with 1.03 Unit/g Fw in control plants. -1,3-Glucanase activity of plants that treated with CTB and 0.5% TNB increased significantly (p ≤ 0.05) compared with that in control plants. GPOD activity increased by 4.78 Unit/g Fw at CTB and 0.5% TNB, whereas GPOD activity in cucumber roots was 2.56 Unit/g Fw in control plants. Nguyen et al. (2013b) suggested that peroxidase and chitinase activities in cucumber leaves were always significantly higher in plants that were treated with TNB compared with plants that were not treated with TNB but inoculated with F. solani alone at 7, 14 and 21 days after treatments. Thus, our findings provide evidence that inducing PR proteins by applying plant extracts may strengthen the plants against various biotic stressor. The SDS-PAGE analysis of enzyme extract from the CTB and TNE treated plants inoculated with F. solani expressed one 1 isoform. Conspicuously, CTB and TNB treated plants expressed a more intense isoform banding pattern compared to that in untreated control plants (Fig. 4a). The native PAGE enzyme extract from the CTB and 0.5% TNE treated plants inoculated with F. solani expressed four isoforms (Gp1, Gp2, Gp3, and Gp4), whereas only three isoform (Gp1, Gp2, and Gp4) were observed in control plants. In particular, the isoform (Gp4) expressed by CTB and 0.3 and 0.5% TNE treated plants was more intense than that of other plants (Fig. 4b). Applying spinach and rhubarb leaf extracts induces systemic resistance to anthracnose disease in cucumber caused by Colletotrichum lagenarium (Doubrava et al., 1988). Schneider and Ullrich (1994)
Fig. 4. Pathogenesis-related proteins in cucumber leaves treated with chitosan beads against F. solani. -1,3-glucanase (a) and GPOD (b). T0: F. solani + chitosan beads (CTB) with 0% Terminalia nigrovenulosa extract (TNE); T1: F. solani + CTB with 0.1% TNE; T2: F. solani + CTB with 0.3% TNE; T3: F. solani + CTB with 0.5% TNE.
reported that Reynoutria sachalinensis extract treatments led to an increase in chitinase, -1,3-glucanase, peroxidase, and polyphenol oxidase activities in cucumber and tobacco. Higher levels of GPOD, -1,3-glucanase, and chitinase are effective against various fungal diseases (Chen et al., 2000; Ramamoorthy and Samiyappan, 2001; Saravanakumar et al., 2007). These results substantiate the inhibition of various plant pathogens and disease management by using several biocontrol agents through induced systemic resistance in plants (Van-Loon and Bakker, 2005; Saravanakumar et al., 2007). 4. Conclusion Based on these results, chitosan beads combined with TNP/TNE could be used as a fungicide to control F. solani. However, further studies are needed to establish human safety and to evaluate their fungi-antagonistic effects under field conditions in order for the practical use of TNB as fungicides to proceed. If commercial bio-fungicides are used as biological control agents against soilborne phytopathogens, formulations that improve their antifungal potency and stability must be developed to reduce costs and make them practical for use in the field. Acknowledgment This study was supported by the Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea. References Akhtar, Y., Yeoung, R., Isman, M.B., 2008. Comparative bioactivity of selected extracts from Meliaceae and some commercial botanical insecticides against two noctuid
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Chitosan beads combined with Terminalia nigrovenulosa bark enhance suppressive activity to Fusarium solani Dang-Minh-Chanh Nguyen a,b , Dong-Jun Seo a , Ro-Dong Park a , Byung-Jin Lee c , 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, Viet Nam c Department of Agronomy, Gyeongnam National University of Science and Technology, Chilam-Dong 150, Jinju-City, Republic of Korea
a r t i c l e
i n f o
Article history: Received 27 March 2013 Received in revised form 2 July 2013 Accepted 25 July 2013 Keywords: Terminalia nigrovenulosa Chitosan beads Biocontrol Fusarium solani Pathogenesis-related proteins
a b s t r a c t Chitosan beads (CTB) and Terminalia nigrovenulosa bark (TNB) were prepared using chitosan powder combined with T. nigrovenulosa powder (TNP) and T. nigrovenulosa extract (TNE) to obtain CTB + TNP (0, 1, 3, and 5%) and CTB + TNE (0, 0.1, 0.3, and 0.5%), respectively. The chitosan beads combined with TNB were tested for their antifungal activity against Fusarium solani. In vitro studies indicated that a mixture of 20 chitosan beads and 3–5% TNP inhibited F. solani mycelial growth the greatest. Mixtures of 10–20 chitosan beads and 0.3–0.5% TNE showed the greatest inhibition of F. solani mycelial growth. Total inhibition of F. solani mycelial growth was achieved with CTB and 5% TNP and CTB and 0.5% TNE after a 6 day incubation. Induction of pathogenesis-related proteins such as chitinase, -1,3-glucanase, and guaiacol peroxidase were tested in cucumber seedlings. Enzyme accumulation was significantly higher in CTB and 0.5% TNE treated cucumber plants compared to that in control plants. We effectively demonstrated the biological activities of the CTB and TNP and CTB and TNE treatments. These results suggest that applying chitosan beads combined with TNB could be useful to control soil-borne diseases of cucumber seedlings. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Diseases caused by Fusarium solani are a limiting factor in plant production, and yield quantity. F. solani causes the death of young and adult plants, with consequent economic losses (Kim and Jee, 1998). Several effective fungicides have been recommended for use against pathogens, but they are not considered long-term solutions due to expense, exposure risk, fungicide residue, and environmental hazard concerns. Therefore, alternative control methods that can be safely and effectively used are urgently required (Kavino et al., 2008). Applying plant extracts and biocontrol agents is eco-friendly and effective against many plant pathogens (Kagale et al., 2004). A number of plant species possess natural substances that are toxic to many pests and pathogens (Akhtar et al., 2008; Harish et al., 2008). These pesticides are generally considered non-persistent under field conditions, as they are readily transformed by light, oxygen, or microorganisms into less toxic products. Therefore, fewer residues are expected from those products (Akhtar et al., 2008). Up to date, various plant extracts exert different levels of antifungal activity in vitro against phytopathogenic fungi (Soylu et al.,
∗ Corresponding author. Tel.: +82 62 530 3960; fax: +82 62 530 2139. E-mail address: [email protected] (W.-J. Jung). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.07.060
2006; Jasso de Rodríguez et al., 2005; Bajwa and Iftikhar, 2005). Chitosan is obtained by partial deacetylation of chitin (Muzzarelli, 2009; Jayakumar et al., 2010). Chitosan has been used as a swollen bead support for preparing immobilized enzymes and conjugate drug carriers (Juang et al., 2001; Yu et al., 2011). Chitosan as fungicides are effective in inhibiting spore germination, germ tube elongation and mycelial growth of fungal phytopathogens, such as Phytophthora capsici and Alternaria solani (Xu et al., 2007), Fusarium (Eweis et al., 2006), and Alternaria kikuchiana and Physalospora piricola (Meng et al., 2010). Additionally, combinations of chitosan and Cornus officinalis seed extract have been showed high antifungal activities, especially against Rhizoctonia solani and F. solani (Seo et al., 2013). Plants in the genus Terminalia, family Combretaceae, comprising some 250 species, are widely distributed in tropical areas of the world (Fabry et al., 1998). A methanol extract of Terminalia arjuna and T. superba bark shows nematicidal and antifungal activities against Haemonchus contortus, Trychophyton rubrum, and Microsporum audouinii (Bachaya et al., 2009; Kuete et al., 2010). Additionally, the presence of phenolic acids such as gallic acid, ellagic acid, and corilagin acid is one of the reasons for the antifungal nature of the Terminalia chebula Retz (Rangsriwong et al., 2009). In our previous studies, Terminalia nigrovenulosa bark (TNB) showed strong biocontrol properties against F. solani (Nguyen et al.,
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2013b) and Meloidogyne incognita (Nguyen et al., 2013a) in vitro. In this study, chitosan beads were used as a carrier to immobilize the agent showing both antifungal and nematicidal activities of TNB. Applying TNB to swollen chitosan beads has not been studied extensively. This study was designed to investigate the activities of TNB combined with swollen chitosan beads against the phytopathogens F. solani. 2. Materials and methods 2.1. Materials Chitosan powder [90% deacetylation, 10 cPs (in 0.5% acetic acid + 0.5% chitosan solution, at 20 ◦ C)] was purchased from Keumho Chemical Co., Ltd. (Seoul, Korea). TNB was collected from DakLak province, Vietnam. F. solani KACC 40384 was obtained from the Korea Agricultural Culture Collection (Suwon, Korea). All other chemicals were of analytical grade. 2.2. Preparation of chitosan beads (CTB) combined with T. nigrovenulosa powder (TNP) and CTB combined with T. nigrovenulosa extract (TNE)
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randomized design under chamber conditions. All procedures were performed under sterile conditions. 2.5. Activity measurement of pathogenesis-related (PR) proteins At 7 days after pathogen inoculation, leaves sample was homogenized with liquid nitrogen in a pre-chilled mortar and pestle. 200 mg of sample was homogenized with 1 mL of 50 mM Tris buffer (pH 6.7), and the homogenate was centrifuged at 10,000 × g for 20 min at 4 ◦ C. The supernatant was used as a crude enzyme extract for assaying chitinase, -1,3-glucanase and guaiacol-peroxidase (GPOD). Protein concentration was determined according to the method described by Bradford (1976) using bovine serum albumin as a standard. Chitinase activity was assayed by measuring the reducing end group, GlcNAc (N-acetyl--d-glucosamine), produced by colloidal chitin (Lingappa and Lockwood, 1962). 1,3-glucanase activity was assayed by measuring the amount of the reducing end group, glucose, produced from laminarin (Yedidia et al., 2000). GPOD activity was assayed by measuring the amount of the reducing end group produced using the method described by Jinmin and Bingru (2001). 2.6. Activity staining of PR proteins
TNB was cut into 0.5–1.0 cm pieces and powdered using a mortar and pestle to obtain TNP. The TNB was also cut into 2–3 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) and then lyophilized to obtain the TNE. The mixtures of CTB and TNP were 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 TNP, respectively. Similarly, mixtures of CTB and TNE were 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 TNE was added, respectively. The resulting viscous solutions of CTB and TNP and CTB and TNE were de-gassed under vacuum and dropped into 200 mL of an alkali coagulating solution (H2 O–MeOH–NaOH = 4:5:1, w/w/w) to prepare highly swollen 3.5 mm average diameter spherical beads (Mitani et al., 1991). The chitosan beads were collected and thoroughly washed with distilled water. 2.3. Testing the antifungal spectrum of TNB using Petri dish Antifungal assays were performed as previously described by Soylu et al. (2006) with slight modifications. The antifungal spectrum was determined by placing a 4 mm-diameter agar disk cut from the edge of an actively growing colony of the test pathogen onto the center of a new Petri dish containing potato dextrose agar medium. Subsequently, the 5, 10, and 20 CTBs of each concentration were placed carefully around a F. solani colony on a PDA plate, respectively. Each combination was tested using three replicates. Zones of inhibition were measured at 2, 4, and 6 days after incubation in the dark at 25 ◦ C. The test was conducted twice. 2.4. Antifungal activity of TNB for disease control Cucumber (C. sativus L. Asia Unchun F1) seeds were sown in steam sterilized soil in 5 cm clay pots, and single seedlings were transplanted into each of the pots containing 200 g of sterile soil 1 week after germination. All seedlings were kept in a growth chamber under a 16/8 h light/dark photoperiod, a 26/20 ◦ C day/night and 60–70% relative humidity. The plants were inoculated with spore suspensions (2 × 105 spores/mL) of F. solani. Subsequently, the cucumber plants were treated with 20 beads of chitosan and TNE (0, 0.1, 0.3, and 0.5%). Three replications were maintained for each treatment, and each replication consisted of three pots in a
Thirty micrograms of enzyme extracts was loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE gels to detect -1,3-glucanase and GPOD activity, respectively. For active staining of -1,3-glucanase after 12% (w/v) SDS-PAGE, a gel was prepared by dipping it in 0.15% triphenyltetrazolium chloride solution containing 1 N NaOH followed by heating in an oven until red bands appeared (Pan et al., 1989). The gels were soaked in 50 mM Tris buffer (pH 6.8) for 10 min to actively stain GPOD after 10% (w/v) native PAGE, and then they were incubated with 0.46% (v/v) guaiacol and 13 mM H2 O2 in the same buffer until red bands appeared. The gels were fixed in water/methanol/acetic acid (6.5:2.5:1, v/v) (Caruso et al., 1999). 2.7. Statistical analysis Data on the effect of the treatments on the growth of pathogens and activity of enzymes in cucumber plants were compared using Tukey’s Studentized range (HSD) test, with a p ≤ 0.05 indicating statistical significance. All data were analyzed using the Statistical Analysis System 9.1 (SAS Institute, Cary, NC, USA) and are presented as mean ± standard deviation. 3. Results and discussion In this study, mixtures of chitosan beads and TNB inhibited growth of F. solani. F. solani mycelia grew slowly on PDA plates containing CTB and TNP (Fig. 1a–c). In particular, the mycelial disk of F. solani treated with 20 CTB and 3% or 5% TNP did not grow for 2, 4, or 6 days (Fig. 1c, T2 and T3). Inhibition of 5 CTB with all tested concentrations against F. solani was only moderate (Fig. 1a). Briefly, mycelial growth was 46.3, 28.7, 18.7, and 18.3 mm at 0, 1, 3, and 5% of TNP, respectively for a 6 day treatment with the 5 CTB (Fig. 1d). Mycelial growth was 46.3, 15.0, 7.7, and 7.7 mm at CTB and 0, 1, 3, and 5% TNP with 10 CTB, respectively after 6 days of treatment (Fig. 1e). The 20 CTB managed to inhibit the mycelial growth of F. solani completely (Fig. 1f). Similary, the results of tests with CTB and TNE revealed that the inhibition of the 5 CTB and TNE (0, 0.1, 0.3, and 0.5%) against F. solani was only moderate (Fig. 2a and d). The 10 CTB and 0.3 or 0.5% TNE significantly inhibited mycelial growth after 4 days of treatment (Fig. 2b and e). The 20 CTB and 0.3 or 0.5% TNE inhibited mycelial growth completely after 6 days of treatment (Fig. 2c and f).
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Fig. 1. Antifungal activity of chitosan beads (CTB) combined with Terminalia nigrovenulosa powder (TNP) against F. solani at 2, 4, and 6 day after incubation. T0: CTB + 0% TNP, T1: CTB + 1% TNP, T2: CTB + 3% TNP, and T3: CTB + 5% TNP. Mycelial growth of F. solani on potato dextrose agar (PDA) treated with five (a and d), ten (b and e), and 20 chitosan beads (c and f). Different letters above the standard error bars indicate a significant difference at each observation time based on Tukey’s Studentized range at p ≤ 0.05.
A concentration of 10% zimmu leaf extract is highly effective for reducing mycelial growth (by 87%) of Alternaria solani (Latha et al., 2009). Kuete et al. (2010) suggested that a methanol extract of Terminalia superba has antifungal activities against Trychophyton rubrum and Microsporum audouinii. Bhutta et al. (2001) tested 32 different seeds and found that seeds from Coriander sativum and Memoranda charata revealed inhibitory effects at 0.5–1.0% concentrations against Alternaria alternata and F. solani. The presence of phenolic acids such as gallic acid, ellagic acid, and corilagin acid are one of the reasons responsible for the antifungal nature of T. chebula Retz (Rangsriwong et al., 2009). Bianchi et al. (1997) reported that the use of garlic micronized powder inhibited mycelial growth of F. solani and R. solani. Eksteen et al. (2001) tested 11 plant extracts against F. oxysporum and R. solani by the agar
dilution method and inhibited mycelial growth comparable to that obtained using carbendazim and difenconazole. In this study, the degree of inhibition varied significantly (p ≤ 0.05) according to the TNB concentrations. Thus, we suggest that in vitro antifungal activity of chitosan combined with TNB was dose-dependent because antifungal activity increased with increasing concentration of TNB. Fig. 3 shows that the mixtures of chitosan beads and TNE significantly inhibited the root system of cucumber plants caused by F. solani. Control plants were treated with CTB but without TNE, which caused complete stunting and root-rot infection (Fig. 3, T0, arrow). In contrast, plants treated with a mixture of CTB and TNE showed root system growth and development normally. In particular, plants treated with mixtures of chitosan beads and TNE (0.5%) showed well developed root systems and leaves (Fig. 3, T3). These
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Fig. 2. Antifungal activity of chitosan beads (CTB) combined with Terminalia nigrovenulosa extract (TNE) against F. solani at 2, 4, and 6 day after incubation. T0: CTB + 0% TNE, T1: CTB + 0.1% TNE, T2: CTB + 0.3% TNE, and T3: CTB + 0.5% TNE. Mycelial growth of F. solani on potato dextrose agar (PDA) treated with five (a and d), ten (b and e), and 20 chitosan beads (c and f). Different letters above the standard error bars indicate a significant difference at each observation time based on Tukey’s Studentized range at p ≤ 0.05.
results agree with those of Lee et al. (1995), who investigated the antigrowth and cytotoxic effects of gallic acid and chebulinic acid in a Terminalia chebula extract. Nguyen et al. (2013b) also showed that treatment with 1000 ppm of TNB resulted in 84% suppression of Fusarium root rot, while control plants were not treated with TNB but inoculated with F. solani alone, which induced up to 100% root rot, with the majority of plants completely stunted. Sundaramoorthy et al. (2012) provided evidence that Pseudomonas fluorescens and Bacillus subtilis effectively inhibit the growth of F. solani in chili plants. One of the mechanisms by which compounds exert their antifungal activity is through the degradation of pathogenic fungi cell walls. Such inhibitors are believed to block the synthesis of chitin
in fungal cell walls (Liu and Zhang, 2004). Chitinolytic enzymes are considered important in the biological control of soil-borne pathogens (Singh et al., 1999; Kim et al., 2008; Latha et al., 2009). PR proteins such as chitinase and -1,3-glucanase protect plants by degrading the walls of pathogenic fungal cells. Kim and Hwang (1994) reported that injecting pepper stems with a pathogenic strain, Phytophthora capsici, induced these PR proteins. Silva et al. (2004) reported that high peroxidase activities occur in pathogeninfected tomato plants during the later stages of infection. In this study, disease resistance in cucumber plants treated with chitosan beads and TNE revealed higher activity and expression of pathogenesis-related proteins against root-rot pathogen. Increased chitinase, -1,3-glucanase and GPOD activities were observed in
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Fig. 3. The efficacy of chitosan beads (CTB) combined with Terminalia nigrovenulosa extract (TNE) on the suppression of root rot diseases by Fusarium solani at 7 days after treatment. T0: F. solani + CTB (0% TNE); T1: F. solani + CTB (0.1% TNE); T2: F. solani + CTB (0.3% TNE); T3: F. solani + CTB (0.5% TNE).
Table 1 Pathogenesis-related protein activity of cucumber leaves in plants treated with Fusarium solani and chitosan beads with different concentrations of Terminalia nigrovenulosa extract (TNE) at 7 days after treatment. Treatment
PR-proteins activity Chitinase (Unit/g Fw)
T0 T1 T2 T3
1.03 1.55 3.38 4.45
± ± ± ±
0.40c 0.50bc 0.73ab 1.06a
-1,3-glucanase (Unit/g Fw) 17.00 23.56 29.04 50.83
± ± ± ±
5.60b 4.07b 5.94b 5.53a
GPOD (Unit/g Fw) 2.56 3.58 4.41 4.78
± ± ± ±
0.60b 0.77ab 0.43a 0.37a
Values shown in each column are means ± standard deviations based on three replicates. The same letters on each column indicate no significant difference based on Tukey’s Studentized range at p ≤ 0.05. T0: F. solani + CTB (0% TNE); T1: F. solani + CTB (0.1% TNE); T2: F. solani + CTB (0.3% TNE); T3: F. solani + CTB (0.5% TNE).
the CTB and TNE treated plants inoculated with F. solani compared to those in untreated control plants at 7 days after inoculation (Table 1). Chitinase activity of plants that were treated with CTB and 0.5% TNB increased significantly to 4.45 Unit/g Fw, compared with 1.03 Unit/g Fw in control plants. -1,3-Glucanase activity of plants that treated with CTB and 0.5% TNB increased significantly (p ≤ 0.05) compared with that in control plants. GPOD activity increased by 4.78 Unit/g Fw at CTB and 0.5% TNB, whereas GPOD activity in cucumber roots was 2.56 Unit/g Fw in control plants. Nguyen et al. (2013b) suggested that peroxidase and chitinase activities in cucumber leaves were always significantly higher in plants that were treated with TNB compared with plants that were not treated with TNB but inoculated with F. solani alone at 7, 14 and 21 days after treatments. Thus, our findings provide evidence that inducing PR proteins by applying plant extracts may strengthen the plants against various biotic stressor. The SDS-PAGE analysis of enzyme extract from the CTB and TNE treated plants inoculated with F. solani expressed one 1 isoform. Conspicuously, CTB and TNB treated plants expressed a more intense isoform banding pattern compared to that in untreated control plants (Fig. 4a). The native PAGE enzyme extract from the CTB and 0.5% TNE treated plants inoculated with F. solani expressed four isoforms (Gp1, Gp2, Gp3, and Gp4), whereas only three isoform (Gp1, Gp2, and Gp4) were observed in control plants. In particular, the isoform (Gp4) expressed by CTB and 0.3 and 0.5% TNE treated plants was more intense than that of other plants (Fig. 4b). Applying spinach and rhubarb leaf extracts induces systemic resistance to anthracnose disease in cucumber caused by Colletotrichum lagenarium (Doubrava et al., 1988). Schneider and Ullrich (1994)
Fig. 4. Pathogenesis-related proteins in cucumber leaves treated with chitosan beads against F. solani. -1,3-glucanase (a) and GPOD (b). T0: F. solani + chitosan beads (CTB) with 0% Terminalia nigrovenulosa extract (TNE); T1: F. solani + CTB with 0.1% TNE; T2: F. solani + CTB with 0.3% TNE; T3: F. solani + CTB with 0.5% TNE.
reported that Reynoutria sachalinensis extract treatments led to an increase in chitinase, -1,3-glucanase, peroxidase, and polyphenol oxidase activities in cucumber and tobacco. Higher levels of GPOD, -1,3-glucanase, and chitinase are effective against various fungal diseases (Chen et al., 2000; Ramamoorthy and Samiyappan, 2001; Saravanakumar et al., 2007). These results substantiate the inhibition of various plant pathogens and disease management by using several biocontrol agents through induced systemic resistance in plants (Van-Loon and Bakker, 2005; Saravanakumar et al., 2007). 4. Conclusion Based on these results, chitosan beads combined with TNP/TNE could be used as a fungicide to control F. solani. However, further studies are needed to establish human safety and to evaluate their fungi-antagonistic effects under field conditions in order for the practical use of TNB as fungicides to proceed. If commercial bio-fungicides are used as biological control agents against soilborne phytopathogens, formulations that improve their antifungal potency and stability must be developed to reduce costs and make them practical for use in the field. Acknowledgment This study was supported by the Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea. References Akhtar, Y., Yeoung, R., Isman, M.B., 2008. Comparative bioactivity of selected extracts from Meliaceae and some commercial botanical insecticides against two noctuid
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