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Diversity and Antimicrobial Activity of Isolated Endophytic Bacteria from Deodeok (Codonopsis Lanceolata) of Different Locations and Ages

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Diversity and Antimicrobial Activity of Isolated Endophytic Bacteria from Deodeok (Codonopsis Lanceolata) of Different Locations and Ages Vol. 7(12), pp. 1015-1028, 19 March, 2013 DOI: 10.5897/AJMR12.1811 ISSN 1996-0808 © 2013 Academic Journals African Journal of Microbiology Research http://www.academicjournals.org/AJMR Full Length Research Paper Diversity and antimicrobial activity of isolated endophytic bacteria from Deodeok (Codonopsis lanceolata) of different locations and ages Young Min Kang1, Chong Kyu Lee2 and Kye Man Cho3* 1Basic Herbal Medicine Research Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM), Daejeon 305-811, Republic of Korea. 2Department of Forest Resources, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea. 3Institute of Fusion Biotechnology, Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea. Accepted 8 March, 2013 Deodeok (Codonopsis lanceolata) were collected from two mountainous areas (Jirisan and Yarongsan) in Korea. Endophytic bacteria were isolated from two, five, and seven-year-old deodeok (Codonopsis lanceolata) roots. The bacterial diversity of deodeok root samples was evaluated by 16S rDNA analysis. In the roots collected from Jirisan, we identified the presence of Bacillus polyfermenticus (JR2-1), Bacillus subtilis (JR2-3 and JR5-5), Bacillus licheniformis (JR2-6), and Bacillus pumilus (JR5-4 and JR7- 2). The various Bacillus genera, such as B. polyfermenticus (YR5-1 and YR7-4), B. licheniformis (YR5-2 and YR7-1), and B. subtilis (YR7-2), were found in the roots derived from Yarongsan. Bacillus pumilus (JR5-4 and JR7-2) isolated from Jirisan exhibited antifungal activity against plant pathogenic fungi. B. subtilis (JR5-5) and B. licheniformis (JR2-6 and JR7-4) showed antifungal activity against Phytophthora capsici, Fusarium oxysporum, and Rhizoctonia solani. This newly demonstrates the antimicrobial activity ability and bacterial diversity of deodeok roots at different ages by comparing two areas that exhibited antimicrobial activity against human pathogens. Key words: Antimicrobial activity, bacterial diversity, deodeok roots, endophytic bacteria. INTRODUCTION Deodeok (Codonopsis lanceolata), belonging to family to treat bronchitis, cough, spasm, and inflammation, and Campanulaceae, has been used as a traditional as a tonic rude drug and are also consumed as a medicinal plant in Korea, Japan, and China. C .lanceolata vegetable (Lee et al., 2002; Kim et al., 2010). Important is a flowering plant and is a variety of bonnet bellflower. diseases affecting deodeok roots are Phytophthora blight, C. lanceolata is called deodeok in Korea and well known Fusarium wilt, and crown rot, caused by Phytophthora for its phytomedical activities, such as antimicrobial, capsici, Fusarium oxysporum, and Pythium ultimum, antioxidant, and immune-defense activities (He et al., respectively (Cho et al., 2002; Cho et al., 2007). 2011). Deodeok roots have been used as an herbal drug Previously, endophytic bacteria have been reported *Corresponding author. E-mail: [email protected]. Tel: +82-55-751-3272. Fax: +82-55-751-3279. 1016 Afr. J. Microbiol. Res. to show antifungal activity against plant pathogenic fungi that could be directed towards the use of endophytic bac- such as, F. oxyporum and Rhizoctonia solani in cotton; teria as bio-control agents against plant pathogens (Asraful- Verticillium dahliae, Verticillium alboatrum, and Rhizoctonia Islam et al., 2010). solani in potato; Sclerotium rolfsii in beans; Verticillium In the present study, we demonstrated the biodiversity longisporum, Rhizoctonia solani, F. oxyporum, and Phy- of endophytic bacteria isolated from deodeok plants root thium ultimum in balloon flower; and Rhizoctonia solani, of different ages collected from Jirisan and Yarongsan Paenibacillus polymyxa, Bacilllus sp., and Pseudomonas areas in Korea. The isolated endophytic bacteria were poae in ginseng (Chen et al., 1995; Cho et al., 2002; evaluated for extracellular hydrolytic enzymes and anti- Graner et al., 2003; Berg et al., 2005; Seo et al., 2010). microbial activities. Endophytes may also be beneficial to the host plant by producing a variety of substances that participate in plant MATERIALS AND METHODS protection (Blanco et al., 2010; Chakraborty et al., 2010). Hence, the compounds produced by endophytic bacteria Isolation of endophytic bacteria from deodeok roots have a potential use in medicine, agriculture, and industry. An investigation of the plant species inhabiting the sou- Endophytic bacteria were isolated from two-, five-, and seven-year- thern mountainous regions of Korea reveals the total old deodeok (C. lanceolata) roots. A total of ten replicated random root samples were collected from two mountainous areas (Jirisan number of plant species mainly found in the mountain and Yarongsan) located on the southern region of South Korea. areas of Jirisan (JR) and Yarongsan (YR). The two repre- Detail geographical information is available at <http://en.wiki- sentative mountains of this region are home to an esti- pedia.org/wiki/ List_of_mountains_of_Korea>. Yarongsan is called a mated 4,000 plant species (Kim and Song, 2011). The same name as Waryongsan, Gyeongsangnam-do in the list. The plant diversity also influences the microbial diversity of roots samples were surface sterilized with 1% sodium hypochlorite forest soils (Kang et al., 2010). for 10 min to remove epiphytic bacteria (Cho et al., 2002; Cho et al., 2007). The external root portion was chopped off (0.5 cm) from the Endophytes are microorganisms that thrive within the margin with a sterile blade. Later, the root tissue was macerated in living tissues of plants. In most cases, the microbial a sterile porcelain mortar along with sterile phosphate buffer (10 relationship with the host plant is symbiotic or mutualistic. mM; pH 7.2). The root extracts were spread on tryptic soy agar The microorganisms are capable of synthesizing bio-ac- (TSA) plates (Difco, NJ, USA) and incubated at 28 and 37°C for 48 h. The bacterial colonies that appeared on TSA were initially screened tive compounds that can be used by the plant as a defense and grouped based on their morphological characteristics (Cho et mechanism against pathogenic fungi and bacteria (Ryu et al., 2002). al., 2000). Endophytes also promote plant growth, sup- press pathogens, detoxify contaminants, solubilize phos- phate, and assist in nitrogen fixation in plants (Zheng et Test microorganisms and growth conditions E. coli al., 2005). Recent studies of endophytic bacteria have focused on their roles within plants with relation to plant Escherichia coli (DH5α) was required for rDNA sequencing and cultured in Luria-Bertani (LB) broth (Difco, NJ, USA) at 37°C. The nutrition, pollutant catabolism, stress or defense responses, plant fungal pathogens, viz., R. solani, Phythium ultimum, Phyto- and invasion of pathogens (Cho et al., 2002). phthora capsici, and F. oxysporum, were provided by the Laboratory Several endophytic bacterial species can be isolated of Phytopathology, Gyeongnam Agricultural Research and from a single plant. Gram-positive and Gram-negative Extension Services, Jinju, Korea. The fungi were cultured on potato endophytic bacteria have been isolated from different dextrose agar (PDA, Difco, NJ, USA) medium at 28°C. Human tissues of various plants (Zinniel et al., 2002). Endophytic pathogens, viz., Escherichia coli KCTC 1682, Salmonella enteritidis KCTC 12456, S. typhimurium KCTC 1925, Shigella flexineri KCTC Gram-positive bacteria, such as Bacillus sp., have been 2008, Shigella sonnei KCTC 2518, B. cereus KCTC 3624, Listeria isolated from cotton, cucumber root, and balloon flower innocula KCTC 3586, Listeria ivanovii KCTC 3444, Listeria mono- root plant (Dujiff et al., 1997; Reva et al., 2002; Lima et cytogenes KCTC 3569 and Staphylococcus aureus KCTC 1621 al., 2005). Paenibacillus polymyxa has been isolated from were collected from the Korean Collection for Type Cultures (KCTC) the Korean ginseng root (Jeon et al., 2003). Endophytic in Biological Resource Center, Daejeon 305-333, Korea. TSA medium was used for the isolation of deodeok endophytic bacteria. Bacillus halmapalus is associated with the rice plant, These bacteria were also cultured on potato dextrose agar (PDA, where the bacterium is harmless to rice plant and pro- Difco, NJ, USA) medium at 28 and 37°C to determine the optimal motes plant growth by penetration (Rosemblueth and temperature for growth. Martimez-Romero, 2006). Continuously, genetic diversity among endophytic populations of crop plants has been Analysis of 16S rDNA of the endophytic bacteria monitored by PCR-based techniques, revealing a range of organisms that belong to several distinct phylogenetic The DNA from endophytic bacteria was extracted with the G-spinTM groups (Garbeva et al., 2001; Cho et al., 2002; Graner et Genomic DNA Extraction Kit (iNtRON Biotechnology, Suwon, al., 2003; Cho et al., 2007; Asraful-Islam et al., 2010). Korea). The 16S rDNA gene was amplified by PCR using endo- The endophytic bacterial diversity of the balloon flower, phytic bacterial DNA as the template and universal primers (877F: 5′-CGGAGAGTTTGATCCTGG-3′, 878R: 5′- Platycodon gradiflorum, was characterized based on 16S TACGGCTACCTTGTTAGCGAC-3′) (Cho et al., 2002; Cho et al., ribosomal DNA (rDNA) gene sequencing. At different ages 2007). The amplification reaction mix also included Super-Therm of the plant, its roots possessed antimicrobial activities DNA polymerase (JMR, Side Cup, Kent, UK), 1.5 mM MgCl2, and 2 Kang et al. 1017 mM dNTP in a final
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