Endophytic Fungi Harbored in Panax Notoginseng
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JGR200_proof ■ 30 July 2016 ■ 1/8 J Ginseng Res xxx (2016) 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 Endophytic fungi harbored in Panax notoginseng: diversity and 66 2 67 3 potential as biological control agents against host plant pathogens 68 4 of root-rot disease 69 5 70 6 1,q 1,q 2 1 1 71 7 Q9 You-Kun Zheng , Cui-Ping Miao , Hua-Hong Chen , Fang-Fang Huang , Yu-Mei Xia , 72 1 1,* 8 You-Wei Chen , Li-Xing Zhao 73 9 1 Key Laboratory of Microbial Diversity in Southwest China of Ministry of Education and Laboratory for Conservation and Utilization of Bio-Resources, 74 10 Yunnan Institute of Microbiology, Yunnan University, Kunming, China 75 11 2 Department of Chemistry and Life Science, Chuxiong Normal University, Chuxiong, China 76 12 77 13 78 14 article info abstract 79 15 80 16 Article history: Background: Endophytic fungi play an important role in balancing the ecosystem and boosting host 81 17 Received 14 August 2015 growth. In the present study, we investigated the endophytic fungal diversity of healthy Panax noto- 82 18 Received in Revised form ginseng and evaluated its potential antimicrobial activity against five major phytopathogens causing root- 31 May 2016 83 rot of P. notoginseng. 19 Accepted 9 July 2016 84 Methods: A culture-dependent technique, combining morphological and molecular methods, was used 20 Available online xxx 85 to analyze endophytic fungal diversity. A double-layer agar technique was used to challenge the phy- 21 86 topathogens of P. notoginseng. 22 Keywords: Results: A total of 89 fungi were obtained from the roots, stems, leaves, and seeds of P. notoginseng, and 87 23 biological control 41 isolates representing different morphotypes were selected for taxonomic characterization. The fungal 88 24 endophytic fungi fungal diversity isolates belonged to Ascomycota (96.6%) and Zygomycota (3.4%). All isolates were classified to 23 genera 89 25 Panax notoginseng and an unknown taxon belonging to Sordariomycetes. The number of isolates obtained from different 90 26 root-rot disease tissues ranged from 12 to 42 for leaves and roots, respectively. The selected endophytic fungal isolates 91 27 were challenged by the root-rot pathogens Alternaria panax, Fusarium oxysporum, Fusarium solani, Phoma 92 28 herbarum, and Mycocentrospora acerina. Twenty-six of the 41 isolates (63.4%) exhibited activity against at 93 29 least one of the pathogens tested. 94 Conclusion: Our results suggested that P. notoginseng harbors diversified endophytic fungi that would 30 95 provide a basis for the identification of new bioactive compounds, and for effective biocontrol of noto- 31 96 32 ginseng root rot. Copyright Ó 2016, The Korean Society of Ginseng, Published by Elsevier. This is an open access article 97 33 under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 98 34 99 35 100 36 101 37 1. Introduction acids [4], and polysaccharides [5], with the most emphasis being on 102 38 saponins. P. notoginseng saponins are considered as the major active 103 39 Panax notoginseng (Burkill) F.H. Chen (Araliaceae) is a perennial ingredients in notoginseng. The saponins display multiple phar- 104 40 herbaceous plant, cultivated mainly in Wenshan, Yunnan, China, macological effects, such as hemostatic [6e9], antioxidant [10,11], 105 41 and has been historically used as both a medicinal herb and food. neuroprotective [12,13], antitumor [14,15], antidiabetic [16,17], and 106 42 Rhizome and roots of P. notoginseng are officially recorded as other activities, and have been extensively used as therapeutic 107 43 notoginseng in the Chinese Pharmacopoeia [1]. About 61 Chinese agents in China. The pronounced efficacies of notoginseng saponins 108 44 patent medicines contain notoginseng, including Yunnan Bai Yao, a have led to the development of several Chinese patent medicines, 109 45 famous hemostatic proprietary herbal remedy. The secondary such as Xuesaitong Capsules/Soft Capsules [18] and Xuesaitong 110 46 metabolites of this plant include saponins [2,3], flavones [2], amino Injection [19]. 111 47 112 48 113 49 * Corresponding author. Key Laboratory of Microbial Diversity in Southwest China of Ministry of Education and Laboratory for Conservation and Utilization of Bio- 114 50 Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, China. Q1 115 E-mail address: [email protected] (L.-X. Zhao). 51 q 116 These authors contributed equally to this work. 52 117 53 http://dx.doi.org/10.1016/j.jgr.2016.07.005 118 54 p1226-8453 e2093-4947/$ e see front matterCopyright Ó 2016, The Korean Society of Ginseng, Published by Elsevier. This is an open access article under the CC BY-NC-ND 119 license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Zheng Y-K, et al., Endophytic fungi harbored in Panax notoginseng: diversity and potential as biological control agents against host plant pathogens of root-rot disease, Journal of Ginseng Research (2016), http://dx.doi.org/10.1016/j.jgr.2016.07.005 JGR200_proof ■ 30 July 2016 ■ 2/8 2 J Ginseng Res 2016;-:1e8 1 In recent years, the demand for notoginseng has been potential of bacterial endophytes of P. notoginseng. However, little is 66 2 increasing. Unfortunately, the yield and quality of notoginseng are known about the fungal community harbored in P. notoginseng. 67 3 severely limited by replanting obstacles [20], and a number of In this study, the diversity of endophytic fungi isolated from 68 4 diseases caused by a plethora of phytopathogens [21]. Among the different tissues of healthy P. notoginseng was evaluated, and the 69 5 diseases, the root-rot disease complex is the most destructive one isolates were screened for their potential antimicrobial activity 70 6 Q2 as it results in yield reduction, no harvest, or low content of active against five major phytopathogens causing root rot of 71 7 ingredients [22]. The reported fungal pathogens causing root rot P. notoginseng. To the best of our knowledge, this is the first report 72 8 include Alternaria panax, Alternaria tenuissima, Cylindrocarpon of the biodiversity, phylogeny, and assessment of biocontrol po- 73 9 destructans, Cylindrocarpon didynum, Rhizoctonia solani, Phytoph- tential of endophytic fungi harbored in P. notoginseng. 74 10 thora cactorum, Phoma herbarum, Fusarium solani, Fusarium oxy- 75 11 sporum [21], and Fusarium flocciferum [23]. Although diverse 2. Materials and methods 76 12 chemical pesticides and some biocontrol methods are used, it is 77 13 difficult to control the root-rot diseases because of the pathogenic 2.1. Isolation of endophytic fungi 78 14 complex. More comprehensive, practical, and ecological methods 79 15 to eradicate the pathogenic diseases in P. notoginseng are urgently Three-year-old healthy P. notoginseng plant samples were 80 16 needed. Furthermore, specific and nonspecific fungi and bacteria collected in October 2013, from a plantation in Wenshan, Yunnan, 81 17 associated with the plant have been found with little information Southwest China. The collected plants were excised into roots, 82 18 about their ecological functions. stems, leaves, and seeds, put into plastic bags, transferred to the 83 19 Microbial communities associated with plants play an impor- laboratory within 24 h, and stored at 4C until the isolation pro- 84 20 tant role in balancing the ecosystem and boosting host growth. cedure of endophytic fungi was carried out. 85 21 Endophytic fungi, which live in healthy plant tissues for at least a The surface sterilization and isolation of fungal endophytes 86 22 part of their life cycle, without causing any noticeable symptoms of were carried out by following the procedures described by Park 87 23 infection or disease, may benefit their host in different ways, such et al [29]. The plant samples were washed thoroughly with running 88 24 as producing bioactive secondary metabolites, promoting germi- tap water to remove soil particles and rinsed six times with distilled 89 25 nation and shoot growth, inducing host plants to tolerate to biotic water. The separated parts (roots, stems, leaves, and seeds; Fig. 1A) 90 26 or abiotic stresses [24e27]. In a previous study, Ma et al [28] were immersed in 75% ethanol solution for 2e3 min, and subse- 91 27 demonstrated high phylogenetic diversity and biocontrol quently transferred to 5.5% sodium hypochlorite solution for 1e2 92 28 93 29 94 30 95 31 96 32 97 33 98 34 99 35 100 36 101 37 102 38 103 39 104 40 105 41 106 42 107 43 108 44 109 45 110 46 111 47 112 48 113 49 114 50 115 51 116 52 117 53 118 54 119 55 120 56 121 57 122 58 123 59 124 60 125 61 126 62 127 63 128 64 Fig. 1. Panax notoginseng, endophytic fungi and bioactivities. (A) Different parts of 3-year-old healthy plant of P. notoginseng; (B) isolation of endophytic fungi; (C) fermentation; 129 65 (D) screening of antagonistic endophytic fungi against host phytopathogens of root-rot disease. 130 Please cite this article in press as: Zheng Y-K, et al., Endophytic fungi harbored in Panax notoginseng: diversity and potential as biological control agents against host plant pathogens of root-rot disease, Journal of Ginseng Research (2016), http://dx.doi.org/10.1016/j.jgr.2016.07.005 JGR200_proof ■ 30 July 2016 ■ 3/8 Y.-K. Zheng et al / Endophytic fungi of P. notoginseng 3 1 min, depending on the different tissues. The surface-sterilized 2.5. In vitro antagonistic activity against host phytopathogens 66 2 samples were rinsed three times with sterile distilled water and 67 3 dried with sterile filter paper.