Endophytic Fungi Harbored in Panax Notoginseng

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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

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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

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. Sterile samples were aseptically Well-grown slant culture of each fungus was used for liquid 68 4 crumbled into small fragments, evenly placed on Petri dishes culture in potato dextrose broth medium (50 mL/250 mL flasks). 69 5 containing potato dextrose agar (PDA) with 100 mg/L ampicillin to The flasks were incubated at 28 1 C on a shaker at 180 rpm Q4 70 6 inhibit bacterial growth. The Petri dishes were incubated at (Fig. 1C) for 7 d. The culture broth was filtered through blotting 71 7 28 1C until fungal growth started. To confirm that the disin- paper and the supernatant was separated. The supernatant was 72 8 fection process was successful, a 0.1-mL aliquot of the water used extracted three times with ethyl acetate (EtOAc). The wet mycelium 73 9 for the last washing step was spotted on PDA plates supplemented was extracted twice with acetone. After removal of acetone in a 74 10 with 100 mg/L ampicillin, and incubated under the same conditions rotary vacuum, the aliquot residue was extracted three times with 75 11 in parallel. Plates that were not detected as contaminated by EtOAc. Combined the EtOAc extracts from supernatant and myce- 76 12 cultivable microorganisms were considered as successfully surface lium, the organic phase was dried over Na2SO4 (anhydrous). The 77 13 disinfected and used for isolation of endophytes [30]. The cultures extracts were then concentrated in a rotary vacuum. The crude 78 14 were monitored every day to monitor the growth of endophytic extracts were stored at 4C before usage. The inhibitory effect of the 79 15 fungi. Each colony that emerged from the fragments was trans- EtOAc extracts obtained from endophytic fungi was tested using the 80 16 ferred to antibiotic-free PDA medium (Fig. 1B), for subculture, and double-layer agar technique [34] (Fig. 1D). The crude extracts were 81 17 brought into pure culture. dissolved in 1.0 mL ethanol as stock solution. A 100-mL volume of 82 18 The pure cultures were recorded and maintained on PDA in the ethanol extract suspension was pipetted into each hole. Test 83 19 active form for further investigation. For long-term storage, the plates were incubated at 28 2C, and the diameters of the inhi- 84 20 fungal colonies were stored in 15% (v/v) glycerol at 80C. All the bition zones were measured after 24 h. The same volume of ethanol 85 21 fungal isolates were deposited in the Yunnan Institute of Microbi- was used as the negative control. 86 22 ology, Yunnan University, Kunming, China. 87 23 3. Results 88 24 2.2. Fungal DNA extraction, PCR amplification and sequencing 89 25 3.1. Phylogenetic analysis of endophytic fungi 90 26 The pure cultures were used for DNA extraction. DNA was 91 92 27 extracted from 0.5e1.0 g mycelia chilled in liquid nitrogen by using A total of 89 endophytic fungi were isolated from the roots, 28 the sodium dodecyl sulfateecetyltrimethyl ammonium bromide stems, leaves, and seeds of healthy notoginseng plants. Forty-one 93 29 method [31]. The internal transcribed spacer (ITS) region was morphotypes were recognizable on the basis of their morpho- 94 30 amplified using primers ITS1 (50-TCCGTAGGTGAACCTGCGG-30) and logical characteristics. One isolate of each morphotype was 95 fi 31 ITS4 (50-TCCTCCGCTTATTGATATGC-30) [32]. The polymerase chain selected for classi cation by phylogenetic analysis. All sequences 96 32 reaction (PCR) was performed in a 50-mL reaction mixture con- were BLAST searched in GenBank (NCBI database). The closest 97 33 taining 1 mL template DNA, 1 mL forward primer (10mM),1 mL reverse matches and the GeneBank accession numbers are listed in 98 34 primer (10mM), 5 mL reaction buffer (10), 4 mL dNTP (each 2.5mM), Table 1. Only isolate PH30454 revealed at least 98% similarity to 99 35 0.5 mL Taq DNA Polymerase (5 U/mL), and 37.5 mL sterile double- known reported sequences. Among 89 endophytic fungi isolates, 100 36 distilled water. The PCR cycling protocol consisted of initial dena- 86 belonged to phylum Ascomycota, and three to Zygomycota. All 101 fi 37 turation at 94C for 4 min, followed by 30 cycles of 94C for 45 s, isolates were classi ed to 12 orders (Capnodiales, Chaetothyriales, 102 38 55C for 45 s, and 72C for 2 min, and a final elongation step of 72C Diaporthales, Eurotiales, Glomerellales, Helotiales, Hypocreales, 103 39 for 10 min. As a negative control, the template DNA was replaced by Microascales, Mucorales, Pleosporales, Sordariales, Xylariales), 23 104 40 sterile double-distilled water. The PCR amplified products were genera (Acremonium, Alternaria, Arthrinium, Aspergillus, Botryoti- 105 41 separated by agarose gel electrophoresis, and sequenced on an ABI nia, Chaetomium, Cladosporium, Colletotrichum, Dictyosporium, 106 42 Prism 3730 sequencer at Sangon Biotech (Shanghai, China). Fusarium, Humicola, Ilyonectria, Mucor, Myrothecium, Penicillium, 107 43 Periconia, Pestalotiopsis, Phialophora, Phoma, Phomopsis, Plectos- 108 44 phaerella, Thielavia, and Trichoderma), in which isolate PH30448 109 45 2.3. Phylogenetic analysis of endophytic fungi belonging to genus Arthrinium was incertae sedis at order level and 110 46 isolate PH30402 was an unknown fungus belonging to 111 fi 47 For strain identi cation, the ITS sequences were compared with Sordariomycetes. 112 48 those in the NCBI database (http://www.ncbi.nlm.nih.gov/) using The phylogenetic tree (Fig. 2) revealed the relationship between 113 fi fi 49 the BLAST search program for the nal identi cation of the fungal the different species of fungal endophytes obtained from different 114 50 endophytes program. A phylogenetic tree was constructed by the parts of P. notoginseng. The most frequently occurring groups were 115 51 Q3 neighbor-joining method using MEGA version 5.0 software, after Hypocreales with 26 isolates (29.2%) and Pleosporales with 19 116 52 pairwise alignments using the CLUSTAL X program, version 1.8 [33]. isolates (21.3%). Hypocreales group included six genera: Acre- 117 53 The stability of the internal branches was assessed with 1,000 monium, Fusarium, Ilyonectria, Myrothecium, Plectosphaerella, and 118 54 bootstrap replications. The sequences of this study were deposited Trichoderma. Pleosporales were integrated by three different 119 e 55 in the GenBank database under the accession numbers: KP714353 genera: Alternaria, Dictyosporium, and Phoma. The groups Capno- 120 56 KP714393 (Table 1). diales, Chaetothyriales, Diaporthales, Glomerellales, Helotiales, 121 57 Microascales, Mucorales, and Xylariales comprised only one taxon 122 58 2.4. Phytopathogens used for antagonistic assays (genus) each (Table 1 and Fig. 2). The results suggested that there 123 59 were diverse endophytic fungi harbored in the cultivated 124 60 A. panax, F. oxysporum, F. solani, Phoma herbarum, and Myco- P. notoginseng. 125 61 centrospora were isolated from rotten root samples of P. notoginseng 126 62 collected from Wenshan in August, 2012. The identification was 3.2. Fungal distribution analysis 127 63 performed by morphological and ITS sequencing methods. The 128 64 pathogenicity was confirmed by experiments with detached roots The number of isolates obtained from different tissues of 129 65 of healthy notoginseng plants (unpublished data). P. notoginseng ranged from 12 to 42 for leaves and roots, 130

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1 Table 1 66 2 Analysis of 89 isolates from Panax notoginseng with the related species and their isolation information 67 3 Phylogenetic group Representative isolate Closest species in GenBank Similarity (%) Tissues of P. notoginseng No. of 68 4 (genus) (accession No.) (accession No.) isolates 69 Roots Stems Leaves Seeds 5 70 6 ASCOMYCETES 71 Capnodiales 7 Cladosporium PH30450 (KP714353) Cladosporium cladosporioides (KJ767066 ) 100 3 8 72 8 PH30409 (KP714354) Cladosporium oxysporum (KJ475816) 100 3 73 9 PH30412 (KP714355) Cladosporium oxysporum (KJ475816) 100 2 74 10 Chaetothyriales 75 Phialophora PH30403 (KP714356) Phialophora mustea (KF850364) 100 1 1 11 76 Diaporthales 12 Phomopsis PH30406 (KP714357) Phomopsis columnaris (KC145883) 100 2 2 77 13 Eurotiales 78 14 Aspergillus PH30427 (KP714358) Aspergillus versicolor (KJ152174) 100 1 3 4 79 15 Penicillium PH30444 (KP714359) Penicillium chrysogenum (LN482541) 100 2 10 80 PH30424 (KP714360) Penicillium crustosum (LN482509) 100 3 16 PH30378 (KP714361) Penicillium cordubense (KJ191427) 100 3 81 17 PH30390 (KP714362) Penicillium lapidosum (KJ676451) 100 2 82 18 Glomerellales 83 19 Colletotrichum PH30420 (KP714363) Colletotrichum gloeosporioides (KF864554) 100 2 2 84 Helotiales 20 85 Botryotinia PH30439 (KP714364) Botryotinia fuckeliana (KF802809) 100 2 2 21 Hypocreales 86 22 Acremonium PH30454 (KP714365) Acremonium implicatum (HQ914932) 98 1 1 87 23 Fusarium PH30410 (KP714366) Fusarium acuminatum (KF887097) 100 1 8 88 24 PH30386 (KP714367) Fusarium oxysporum (KJ026700) 100 3 89 PH30436 (KP714368) Fusarium solani (KF939488) 100 2 2 25 Ilyonectria PH30396 (KP714369) Ilyonectria macrodidyma (HQ703420) 100 2 3 90 26 PH30398 (KP714370) Ilyonectria macrodidyma (HQ703420) 100 1 91 27 Myrothecium PH30382 (KP714371) Myrothecium sp. (HQ631058) 99 2 1 3 92 28 Plectosphaerella PH30464 (KP714372) Plectosphaerella sp. (JX535104) 100 1 1 2 5 93 PH30457 (KP714373) Plectosphaerella sp. (JX535104) 99 1 29 94 Trichoderma PH30435 (KP714374) Trichoderma longibrachiatum (KJ767089) 100 1 6 30 PH30441 (KP714375) Trichoderma koningiopsis (KM079603) 100 2 1 1 95 31 PH30432 (KP714376) Trichoderma spirale (KM079610) 100 1 96 32 Microascales 97 33 Periconia PH30428 (KP714377) Periconia byssoides (KC954160) 99 1 1 98 Pleosporales 34 Alternaria PH30445 (KP714378) Alternaria sp. (KC771455) 100 1 15 99 35 PH30438 (KP714379) Alternaria alternata (KM015489) 100 2 5 100 36 PH30452 (KP714380) Alternaria tenuissima (KF308886) 100 2 2 101 37 PH30425 (KP714381) Alternaria tenuissima (KF308886) 100 1 1 102 PH30442 (KP714382) Alternaria alternata (KM015489) 100 1 38 103 Dictyosporium PH30404 (KP714383) Dictyosporium digitatum (DQ018089) 98 1 1 39 Phoma PH30459 (KP714384) Phoma draconis (GU237795) 100 1 3 104 40 PH30391 (KP714385) Phoma radicina (JQ676200) 99 2 105 41 Sordariales 106 42 Chaetomium PH30461 (KP714386) Chaetomium globosum (KJ728838) 100 1 1 107 Humicola PH30417 (KP714387) Humicola fuscoatra (JN031580) 99 1 1 43 Thielavia PH30430 (KP714388) Thielavia arenaria (JX535015) 99 1 1 108 44 Incertae sedis 109 45 Arthrinium PH30448 (KP714390) Arthrinium arundinis (KF850624) 99 2 2 110 46 Xylariales 111 Pestalotiopsis PH30451 (KP714391) Pestalotiopsis vismiae (JX305715) 100 2 4 47 112 PH30414 (KP714392) Pestalotiopsis uvicola (JN198506 ) 100 2 48 Incertae sedis PH30402 (KP714389) Sordariomycetes sp. (JX244023) 99 2 2 113 49 ZYGOMYCETES 114 50 Mucorales 115 51 Mucor PH30392 (KP714393) Mucor hiemalis (JF299220) 100 3 3 116 No. of total isolates 42 16 12 19 89 52 117 53 118 54 119 55 respectively (Table 1). Forty-two isolates from roots were assigned 3.3. Antagonism assay of fungal endophytes as potential biocontrol 120 56 to 16 genera and an unknown taxon, and the genera Fusarium, agents 121 57 Cladosporium, and Penicillium were the dominant groups. Nineteen 122 58 isolates from seeds belonged to nine genera. Eight genera fungi (16 All EtOAc crude extracts of 41 fungal endophyte strains were 123 59 isolates) were obtained from stems. The Alternaria genus was tested for antagonistic activities against five major fungal patho- 124 60 dominant in seeds and stems. The amount of endophytic fungi from gens that cause root-rot disease of P. notoginseng (Table 2). Twenty- 125 61 leaves was the least, with only 12 isolates belonging to four genera. six of the 41 isolates (63.4%) exhibited activity against at least one of 126 62 In addition, the distribution of endophytic fungi showed significant the pathogens tested. The highest frequency (16 isolates, 39.0%) 127 63 differences in various organs and showed obvious specificity. For was against the pathogen A. panax, followed by F. solani (15 isolates, 128 64 example, the endophytic fungi belonging to Colletotrichum, Ilyo- 36.6 %), F. oxysporum (14 isolates, 34.1%), Phoma herbarum (12 iso- 129 65 nectria, and Mucor were only isolated from roots. lates, 29.3%), and M. acerina (7 isolates, 17.1%), respectively. Five 130

PH30435 71 Trichoderma longibrachiatum (KJ767089) 56 PH30441 1 Trichoderma koningiopsis (KM079603) 66 91 PH30432 2 62 67 3 66 Trichoderma spirale (KM079610) 68 Plectosphaerella sp. (JX535104) 4 PH30464 69 100 5 PH30457 70 72 6 PH30454 71 100 Acremonium implicatum (HQ914932) 7 Fusarium solani (KF939488) 72 8 PH30386 73 99 Fusarium oxysporum (KJ026700) 9 PH30382 74 10 100 Myrothecium sp. (HQ631058) 75 11 PH30396 76 Ilyonectria macrodidyma (HQ703420) 12 99 77 56 PH30398 13 PH30410 78 Fusarium tricinctum (KJ598869) 14 79 99 Fusarium acuminatum (KF887097) 15 80 99 PH30430 16 62 Thielavia subthermophila (JN390827) 81 17 Thielavia arenaria (JX535015) 82 91 PH30461 18 Chaetomium globosum (KJ728838) 83 57 19 95 PH30417 84 Humicola fuscoatra (JN031580) 20 85 PH30436 21 PH30420 86 22 100 Colletotrichum gloeosporioides (KF864554) 87 PH30406 23 88 100 Phomopsis columnaris (KC145883) 24 86 PH30448 89 25 93 Arthrinium arundinis (KF850624) 90 97 26 PH30402 91 87 Sordariomycetes sp. (JX244023) Ascomycota 27 Pestalotiopsis vismiae (JX305715) 92 28 93 Pestalotiopsis uvicola (JN198506) 93 PH30382 29 98 Pestalotiopsis guepinii (KJ934363) 94 30 Pestalotiopsis cocculi (KC837102) 95 31 PH30414 96 Cladosporium cladosporioides (KJ767066) 32 97 100 PH30450 33 PH30412 98 34 Cladosporium oxysporum (KJ475816) 99 PH30409 35 PH30427 100 36 98 Aspergillus versicolor (KJ152174) 101 58 100 66 PH30444 37 Penicillium chrysogenum (LN482541) 102 38 PH30378 103 Penicillium lapidosum (KJ676451) 39 86 104 Penicillium cordubense (KJ191427) 40 PH30390 105 41 92 Penicillium polonicum (KF494148) 106 42 PH30424 107 Penicillium crustosum (LN482509) 43 100 PH30403 108 44 Phialophora mustea (KF850364) 109 Botryotinia fuckeliana (KF802809) 45 99 110 PH30439 50 46 Botrytis cinerea (KM016533) 111 47 92 PH30459 112 48 Phoma draconis (GU237795) 113 PH30391 49 95 Phoma radicina (JQ676200) 114 50 Phoma sp.(KJ882907) 115 PH30442 51 60 116 63 PH30445 52 PH30452 117 53 PH30425 118 93 PH30438 54 Alternaria sp. (KC771455) 119 55 Alternaria tenuissima (KF308886) 120 56 Alternaria alternata (KM015489) 121 93 PH30428 57 Periconia byssoides (KC954160) 122 58 PH30404 123 59 75 Dictyosporium digitatum (DQ018089) 124 60 Mucor hiemalis (JF299220) 125 PH30392 Zygomycota 61 100 Mucor nidicola (HQ913647) 126 62 127 63 0.05 128 64 Fig. 2. Phylogenetic tree based on neighbor-joining analysis of the rDNA internal transcribed spacer sequences of the endophytic fungi obtained from different tissues of Panax 129 65 notoginseng. Signiﬁcant bootstrap values (> 50%) are indicated at the branching points. The tree has been drawn to scale (0.05). 130

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1 Table 2 tissue type studied [37]. Even so, endophytic fungi are still a poorly 66 2 Endophytic fungal antagonists of root-rot disease pathogens investigated group of microorganisms [38], and their complex 67 3 Isolate No. Antagonistic activity1),2) ecological functions remain to be extensively exploited. 68 4 69 Alternaria Fusarium Fusarium Phoma Mycocentrospora P. notoginseng, Panax ginseng, and Panax quinquefolius, have been 5 panax, oxysporum solani herbarum acerina widely used for medicinal plants all over the world. There are a few 70 6 investigations of the endophytic fungi in Panax genus, but most of 71 PH30378 þ þ e 7 PH30382 them have been focused on ginseng (P. ginseng) [29,39 42] and 72 8 PH30386 þ American ginseng (P. quinqefolius) [43]. P. notoginseng, an important 73 9 PH30390 þ Chinese medicine plant, has attracted increasing attention in 74 10 PH30391 medical and chemical fields because of its active ingredients, but 75 PH30392 þ 11 76 PH30396 little in ecological and other research areas because of its narrow 12 PH30398 distribution. 77 13 PH30402 þ In the present study, the diversity, tissue distribution, and 78 14 PH30403 þþ þ biocontrol potential of cultivable endophytic fungi harbored in 79 þ 15 PH30404 P. notoginseng plants were described for the first time. We proposed 80 PH30406 16 PH30409 þþ þ þþþ þ þ that the endophytic fungi would exert certain functions to help the 81 17 PH30410 host survive in plantation against disastrous root-rot diseases. In 82 18 PH30412 þþþþþþ þ the survived and healthy notoginseng plants, the harbored fungal 83 þþ þ þ þþþ þ 19 PH30414 endophytes may give some clue to find effective approaches to 84 PH30417 20 85 PH30420 þþ þþ þþþ control root rot, caused by complex pathogens. Thus, in order to 21 PH30424 þþþ þþ þþ þþ screen for the most promising endophytes, we evaluated the po- 86 22 PH30425 þ tential of all the endophytic isolates as BCAs by challenging five 87 23 PH30427 þ þþ major root-rot pathogens of the host plant, A. panax, F. oxysporum, 88 24 PH30428 F. solani, Phoma herbarum, and M. acerina, whose pathogenicity was 89 PH30430 fi 25 PH30432 þþþ con rmed in our previous work. Among the isolates, crude extracts 90 26 PH30435 þþ of PH30409 and PH30441 showed strong inhibition on all tested 91 27 PH30436 phytopathogens, implicating that the two isolates have the poten- 92 28 PH30438 tial to produce active compounds. Other isolates showed selective 93 PH30439 þþ þ 29 94 PH30441 þþ þþþ þþþ þ þ activity against one or two pathogens. According to the viewpoint 30 PH30442 þ by Zhang et al [36], the mechanisms involved in biocontrol of 95 31 PH30444 þþ þþ þþ þ þ endophytic fungi against phytopathogens include antibiosis, 96 32 PH30445 competition for nutrients and space, induction of defense response, 97 þ 33 PH30448 and mycoparasitism. The endophytic isolates can be used to screen 98 PH30450 þþ þ þ 34 PH30451 þþ their antagonistic activity by fermenting in different media in 99 35 PH30452 þ further work. Other mechanisms should also be investigated in the 100 36 PH30454 þ development of BCAs. For instance, Trichoderma spp. widely used in 101 þ 37 PH30457 agriculture as biofungicides, have shown many ways against phy- 102 PH30459 38 103 PH30461 þþ þþ þþ topathogens, such as producing active compounds, secreting hy- 39 PH30464 drolytic enzymes, and competing for nutrients [44]. In this study, 104 40 isolates belonging to Trichoderma were also obtained and tested as 105 1) Estimated by measuring the diameter of the clear zone of growth inhibition 41 2) Symbols: , no activity; þ, þþ, and þþþ, weak activity, moderate activity, and active endophytic strains. Endphytic fungus Penicillium chrys- 106 42 strong activity, respectively ogenum showed effects on various plant pathogens [45e47]. García 107 43 et al [48] reported that the isolate EEZ10 of Penicillium chrysogenum 108 44 from soil inhibited the growth of Verticillium dahlia in vitro, and is a 109 45 isolates, PH30409, PH30412, PH30414, PH30441, and PH30444, possible biological control agent for Verticillium disease [48]. The 110 46 were found to inhibit all pathogens tested, implicating they have a sequence of isolate PH30444 was 100% similar to that of Penicillium 111 47 broad spectrum of antagonistic activity. Isolates, PH30409, chrysogenum, and its crude extract also exhibited inhibition against 112 48 PH30414, PH30420, PH30424, and PH30441 exhibited relatively all tested host phytopathogens. Recently, we found that some 113 49 strong inhibitory effects against the pathogens F. oxysporum, endophytic isolates can inhibit pathogens of notoginseng root-rot 114 50 F. solani, and Phoma herbarum. The broad spectrum or strong diseases by mycoparasitism and producing active secondary me- 115 51 inhibitory activities of these strains against the host plant patho- tabolites and volatile organic compounds [49e54]. These findings 116 52 gens suggest that these endophytic fungi may be potential candi- showed that endophytic fungi from P. notoginseng are potential 117 53 dates for the production of bioactive compounds, and have the BCAs for the host plant. 118 54 potential as biological control agents of root-rot disease of The culture-based study and molecular identification showed 119 55 P. notoginseng. In other virulence tests with detached healthy that the total fungal diversity was relatively high, and the over- 120 56 notoginseng roots, isolates PH30409 and PH30412 were found to be whelming majority belonged to Ascomycota (96.6% of the total 121 57 able to cause root rot (unpublished data), although they showed number of isolates). Only a single Zygomycota group was obtained. 122 58 antagonism to tested pathogens. This finding is consistent with a previous study in Cannabis sativa L. 123 59 [26]. In 12 morphologically distinct groups, Hypocreales (29.2%) 124 60 4. Discussion and Pleosporales (21.3%) were dominant orders among the endo- 125 61 phytic fungi of different tissues of P. notoginseng. This result was 126 62 Endophytic fungi have been considered as a promising source similar to the previous studies in many medicinal plants [26,55]. 127 63 for the development of biological control agents (BCAs) against The endophytic fungi associated with P. notoginseng comprised a 128 64 phytopathogens, because they exhibit the beneficial functions for number of dominant groups, such as Alternaria, Fusarium, and 129 65 host plants [24e27,35,36], and are ubiquitous within almost every Penicillium. All these genera have been previously isolated as 130

Y.-K. Zheng et al / Endophytic fungi of P. notoginseng 7

1 endophytes, not only from other Panax species [29,39,40,42], but planted in Southwest China. The fungal endophytes serve as great 66 2 also from a wide range of plant hosts in the temperate and tropical promise not only as BCAs against the known and emerging phy- 67 3 zones [26,56,57]. Although, genera Alternaria, Cladosporium, Colle- topathogens of P. notoginseng, but also as a resource of biologically 68 4 totrichum, and Fusarium were found as endophytes in all three active novel secondary metabolites [65]. Three endophytic fungal 69 5 Panax species (P. ginseng, P. notoginseng, and P. quinquefolium) isolates, PH30444, PH30414, and PH30441, are potential BCAs 70 6 [29,39e43], other genera Arthrinium, Botryotinia, Chaetomium, against A. panax, F. oxysporum, F. solani, Phoma herbarum, and 71 7 Dictyosporium, Humicola, Ilyonectria, Mucor, Periconia, Pestalo- M. acerina, and possibly other pathogens. Further studies are 72 8 tiopsis, and Thielavia, have not been obtained from other Panax required to clarify the biocontrol active metabolites and the control 73 9 plants, suggesting that P. notoginseng harbors specific and diverse efficiency of the potential endophytic isolates in both pot and field 74 10 fungi due to the unique soil, climatic conditions of Wenshan region experiments. A full understanding of endophytic community 75 11 and the approaches taken in agro-management. structure, dynamics, and functions, and the shaping factors will be 76 12 Some isolated taxa showed preference for specific tissues types. helpful in selecting pesticide, fertilizer, and other approaches for 77 13 Previous studies in other plants have also indicated that endo- notoginseng planting. 78 14 Q5 phytes exhibit tissues specificity. As expected, tissue specificity of 79 15 fungal endophytes was demonstrated in our study. Particular en- 80 Conflict of interest 16 dophytes were only found in one tissue, which suggests that certain 81 17 species survive within the specific chemistry or texture of different 82 The authors declare that there are no conflicts of interests. 18 tissues [57]. The diversity and species richness of endophytic fungi 83 19 were higher in the underground part (roots) than that in the above- 84 20 ground parts (stems, leaves, and seeds). Some similar results have Acknowledgments 85 21 been reported in the previous studies [55,57]. The possible reasons 86 22 are that soil-borne fungi are typically more prevalent and diversi- This work was partly supported by the grants from the National 87 23 fied than those that infect aerial plant tissues [55], and there are Natural Science Foundation of China (Nos. 31100009 and 88 24 abundant nutriments in roots of P. notoginseng that are suitable for 41361075), Yunnan Natural Science Foundation (No. 2013FA015), 89 25 the growth of fungi. The relative abundance of each fungus iden- and Foundation of Yunnan Educational Committee (No. 90 26 tified in P. notoginseng reflects an unequal distribution of isolate ZD2013008). 91 27 richness among species. Similar results have been found in other 92 28 plants, such as Stellera chamaejasme and Taxus globose [55,57]. References 93 29 Planteendophytic fungus interactions have been extensively 94 30 investigated. Most of the symbionts may have helped for host [1] He HM, Cui L. Pharmacopoeia of the People’s Republic of China. Beijing: 95 31 plants. It has also been confirmed that the pathogeniceendophytic Chinese Medical Science Press; 2010. 96 [2] Yang WZ, Hu Y, Wu WY, Ye M, Guo DA. Saponins in the genus Panax L. 32 lifestyles of some fungi are interchangeable [58,59]. These phe- (Araliaeae): a systematic review of their chemical diversity. 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