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Diversity, Distribution, and Antagonistic Activities of Rhizobacteria of Panax

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Diversity, Distribution, and Antagonistic Activities of Rhizobacteria of Panax JGR108_proof ■ 5 June 2015 ■ 1/8 J Ginseng Res xxx (2015) 1e8 55 Contents lists available at ScienceDirect 56 57 Journal of Ginseng Research 58 59 60 journal homepage: http://www.ginsengres.org 61 62 63 Research article 64 65 1 Diversity, distribution, and antagonistic activities of rhizobacteria of 66 2 67 3 Panax notoginseng 68 4 69 1,2,4 2,4 2 2 3 5 Q13 Ze-Yan Fan , Cui-Ping Miao , Xin-Guo Qiao , You-Kun Zheng , Hua-Hong Chen , 70 6 You-Wei Chen 2, Li-Hua Xu 2, Li-Xing Zhao 2,*, Hui-Lin Guan 1,** 71 7 72 8 1 School of Energy and Environment Science, Yunnan Normal University, Kunming, PR China 2 73 9 Key Laboratory of Microbial Diversity in Southwest China of Ministry of Education and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming, PR China 74 10 3 Department of Chemistry and Life Science, Chuxiong Normal University, Chuxiong, PR China 75 11 76 12 77 13 article info abstract 78 14 79 15 Article history: Background: Rhizobacteria play an important role in plant defense and could be promising sources of 80 16 Received 9 February 2015 biocontrol agents. This study aimed to screen antagonistic bacteria and develop a biocontrol system for 81 17 Received in Revised form root rot complex of Panax notoginseng. 82 2 May 2015 18 Methods: Pure-culture methods were used to isolate bacteria from the rhizosphere soil of notoginseng Accepted 9 May 2015 83 plants. The identification of isolates was based on the analysis of 16S ribosomal RNA (rRNA) sequences. 19 Available online xxx 84 20 Results: A total of 279 bacteria were obtained from rhizosphere soils of healthy and root-rot notoginseng 85 plants, and uncultivated soil. Among all the isolates, 88 showed antagonistic activity to at least one of 21 Keywords: 86 three phytopathogenic fungi, Fusarium oxysporum, Fusarium solani, and Phoma herbarum mainly causing 22 antagonistic activities 87 23 diversity root rot disease of P. notoginseng. Based on the 16S rRNA sequencing, the antagonistic bacteria were characterized into four clusters, Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetesi. The genus 88 24 Panax notoginseng rhizobacteria Bacillus was the most frequently isolated, and Bacillus siamensis (Hs02), Bacillus atrophaeus (Hs09) 89 25 showed strong antagonistic activity to the three pathogens. The distribution pattern differed in soil types, 90 26 genera Achromobacter, Acidovorax, Brevibacterium, Brevundimonas, Flavimonas, and Streptomyces were 91 27 only found in rhizosphere of healthy plants, while Delftia, Leclercia, Brevibacillus, Microbacterium, Pantoea, 92 28 Rhizobium, and Stenotrophomonas only exist in soil of diseased plant, and Acinetobacter only exist in 93 29 uncultivated soil. 94 30 Conclusion: The results suggest that diverse bacteria exist in the P. notoginseng rhizosphere soil, with 95 fi 31 differences in community in the same eld, and antagonistic isolates may be good potential biological 96 control agent for the notoginseng root-rot diseases caused by F. oxysporum, Fusarium solani, and Panax 32 97 33 herbarum. Copyright Ó 2015, The Korean Society of Ginseng, Published by Elsevier. All rights reserved. 98 34 99 35 100 36 101 37 1. Introduction of coronary heart disease and cardiovascular disease [2]. Roots of 102 38 P. notoginseng have been used as a variety of raw materials in 103 39 Panax notoginseng F. H. Chen, known as Sanqi or Tianqi in Chi- Chinese medicinal products in China [3]. It has been mainly culti- 104 40 nese, is a well-known traditional Chinese medicine [1], widely used vated for 400 years in the Southwest regions of China, especially in 105 41 for promotion of blood circulation, removal of blood stasis, induc- Wenshan, Yunnan Province [4]. 106 42 tion of blood clotting, relief of swelling, alleviation of pain, and cure 107 43 108 44 109 45 110 * 46 Corresponding author. Li-Xing Zhao, Key Laboratory of Microbial Diversity in Southwest China of Ministry of Education and Laboratory for Conservation and Utilization of 111 47 Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, PR China. Q1 ** 112 48 Corresponding author. Hui-Lin Guan, School of Energy and Environment Science, Yunnan Normal University, Kunming 650092, PR China. E-mail addresses: [email protected] (L.-X. Zhao), [email protected] (H.-L. Guan). 113 49 114 50 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) 115 51 which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 116 4 These two authors contributed equally to this work. 52 117 53 1226-8453/$ e see front matter Copyright Ó 2015, The Korean Society of Ginseng, Published by Elsevier. All rights reserved. 118 54 http://dx.doi.org/10.1016/j.jgr.2015.05.003 119 Please cite this article in press as: Fan Z-Y, et al., Diversity, distribution, and antagonistic activities of rhizobacteria of Panax notoginseng, Journal of Ginseng Research (2015), http://dx.doi.org/10.1016/j.jgr.2015.05.003 JGR108_proof ■ 5 June 2015 ■ 2/8 2 J Ginseng Res 2015;-:1e8 1 P. notoginseng should be grown in the field for at least 3 y to obtained without planting notoginseng at the same field. All the 66 2 obtain high-quality raw roots [5]. However, the long period soil samples were placed into sterile plastic bags, transferred to the 67 3 planting conditions make P. notoginseng vulnerable to attacks by laboratory in 24 h, and kept at 4C before treatment. 68 4 many soil-borne pathogens including fungi, bacteria, and nema- Bacterial isolation were developed using serial dilution spread 69 5 todes [5e14]. Soil-borne pathogens of P. notoginseng have been plate method. Ten grams of soil was mixed with 90 mL of sterile 70 6 reported by fungi including Fusarium oxysporum, Fusarium solani, phosphate buffered saline (PBS, pH 7.4) and stirred for 30 min at 71 7 Phoma herbarum, Alternaria tenuis, Alternaria panax, Cylindrocarpon 200 rpm. The soil suspension was left to stand for 10 min at room Q2 72 8 destructans, Cylindrocarpon didynum, Phytophthora cactorum, temperature to allow settling of large particles, tenfold serial 73 À À 9 Rhizoctonia solani, and by bacterial pathogens including Pseudo- diluted in PBS (from 10 2 to 10 5). Then, 80 mL of the first to fourth 74 10 monas sp., Ralstonia sp., and by parasitic nematodes, such as Dity- and fifth diluents were transferred to petri dishes with LB agar 75 11 lenchus sp., Rhabditis elegans, and Meloidogyne spp. [15,16]. In this medium (10.0 g peptone, 5.0 g yeast extract, 10.0 g NaCl, and 13.0 g 76 12 case, the control of soil-borne diseases mainly relies on chemical agar, 1.0 L distilled water, pH 7.2) and nutrition agar (NA) medium 77 13 pesticides, fungicides, and crop rotation. Chemical pesticides and (3.0 g beef extract, 5.0 g peptone, 5.0 g NaCl, 13.0 g agar, 1.0 L 78 14 fungicides are less effective on the soil-borne diseases, and lead to distilled water, pH 7.0). The plates were incubation at 28C, and 79 15 reduction of P. notoginseng quality. Meanwhile, pesticides may be bacterial colonies were selected and purified according to their 80 16 toxic to crops, humans, animals [17,18]. However, a 15e20 y morphological characteristics. 81 17 replanting interval leads to the lack of appropriate fields, resulting 82 18 in searching for a new field or/and transferring to a less appropriate 83 2.2. Screening of antagonistic bacteria against fungal pathogens 19 field to grow P. notoginseng. 84 20 It is obvious that pesticides and less appropriate cultivation soil 85 Three fungal pathogens F. oxysporum, F. solani, and P. herbarum 21 are not suitable to control the qualities of P. notoginseng required by 86 were isolated from the rotten root of P. notoginseng, and their 22 the good agriculture practice (GAP). Friendly approaches are ur- 87 pathogenicity was verified [15,16]. The target fungi were cultured 23 gently needed to effectively manage or solve the questions. Bio- 88 on potato dextrose agar (PDA) medium (200.0 g fresh potato, 20.0 g 24 logical control, a bioeffector method with other living organisms to 89 starch, 13.0 g agar, 1.0 L distilled water, pH not adjusted). The 25 control pests (insects, mites, weeds, and plant diseases) [19], has 90 antagonism of all bacterial isolates was checked with respect to the 26 been considered as effective approaches. Soil bacteria, especially 91 ability to suppress fungal growth. Antifungal bioassay was per- 27 rhizospheric ones with antagonistic properties, demonstrate bio- 92 formed with the dual culture and agar well diffusion plate on PDA. 28 logical control effectiveness to some plant diseases, and are the 93 In dual culture tests, a 5-mm mycelial disk of pathogenic fungus, 29 most potential for development of biological control agents (BCAs) 94 collected from the edge of actively growing colonies, was placed 30 [20e29]. However, little is known about the bacterial diversity, 95 into the center of plates containing fresh PDA. Bacterial isolates 31 distribution, and ecological effects in the cultivation soil of 96 were grown around the target fungus with a distance of 3.0 cm 32 P. notoginseng. In this study, we developed the investigation of 97 (Fig. 1A, 1B). The dual culture plates were incubation at 28C, and 33 rhizobacteria of 3-y-old P. notoginseng from Wenshan, Yunnan 98 checked every 12 h after inoculation. All treatments were tested in 34 Province, by culture-dependent methods.
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