[국내논문]Endophytic fungi harbored in Panax notoginseng: diversity and potential as biological control agents against host plant pathogens of root-rot disease원문보기
Zheng, You-Kun
(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)
,
Miao, Cui-Ping
(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)
,
Chen, Hua-Hong
(Department of Chemistry and Life Science, Chuxiong Normal University)
,
Huang, Fang-Fang
(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)
,
Xia, Yu-Mei
(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)
,
Chen, You-Wei
(Key Laboratory of Microbial Diversity in Southwest China of Ministry of Education and Laboratory for Conservation and Uti)
,
Zhao, Li-Xing
Background: Endophytic fungi play an important role in balancing the ecosystem and boosting host growth. In the present study, we investigated the endophytic fungal diversity of healthy Panax notoginseng and evaluated its potential antimicrobial activity against five major phytopathogens causing roo...
Background: Endophytic fungi play an important role in balancing the ecosystem and boosting host growth. In the present study, we investigated the endophytic fungal diversity of healthy Panax notoginseng and evaluated its potential antimicrobial activity against five major phytopathogens causing root-rot of P. notoginseng. Methods: A culture-dependent technique, combining morphological and molecular methods, was used to analyze endophytic fungal diversity. A double-layer agar technique was used to challenge the phytopathogens of P. notoginseng. Results: A total of 89 fungi were obtained from the roots, stems, leaves, and seeds of P. notoginseng, and 41 isolates representing different morphotypes were selected for taxonomic characterization. The fungal isolates belonged to Ascomycota (96.6%) and Zygomycota (3.4%). All isolates were classified to 23 genera and an unknown taxon belonging to Sordariomycetes. The number of isolates obtained from different tissues ranged from 12 to 42 for leaves and roots, respectively. The selected endophytic fungal isolates were challenged by the root-rot pathogens Alternaria panax, Fusarium oxysporum, Fusarium solani, Phoma herbarum, and Mycocentrospora acerina. Twenty-six of the 41 isolates (63.4%) exhibited activity against at least one of the pathogens tested. Conclusion: Our results suggested that P. notoginseng harbors diversified endophytic fungi that would provide a basis for the identification of new bioactive compounds, and for effective biocontrol of notoginseng root rot.
Background: Endophytic fungi play an important role in balancing the ecosystem and boosting host growth. In the present study, we investigated the endophytic fungal diversity of healthy Panax notoginseng and evaluated its potential antimicrobial activity against five major phytopathogens causing root-rot of P. notoginseng. Methods: A culture-dependent technique, combining morphological and molecular methods, was used to analyze endophytic fungal diversity. A double-layer agar technique was used to challenge the phytopathogens of P. notoginseng. Results: A total of 89 fungi were obtained from the roots, stems, leaves, and seeds of P. notoginseng, and 41 isolates representing different morphotypes were selected for taxonomic characterization. The fungal isolates belonged to Ascomycota (96.6%) and Zygomycota (3.4%). All isolates were classified to 23 genera and an unknown taxon belonging to Sordariomycetes. The number of isolates obtained from different tissues ranged from 12 to 42 for leaves and roots, respectively. The selected endophytic fungal isolates were challenged by the root-rot pathogens Alternaria panax, Fusarium oxysporum, Fusarium solani, Phoma herbarum, and Mycocentrospora acerina. Twenty-six of the 41 isolates (63.4%) exhibited activity against at least one of the pathogens tested. Conclusion: Our results suggested that P. notoginseng harbors diversified endophytic fungi that would provide a basis for the identification of new bioactive compounds, and for effective biocontrol of notoginseng root rot.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
Groups of fungal phytopathogens also contain large numbers of endophytic taxa. In this study, isolates PH30409 andPH30412 caused root-rot symptoms in experimental infection through detached healthy roots of P. notoginseng (unpublished data). Interestingly, crude extracts of them showed antagonistic activities against the tested phytopathogens, and more experimental infections to confirm the pathogenicity of Cladosporium sp.
제안 방법
5 μL sterile double-distilled water. The PCR cycling protocol consisted of initial denaturation at 94℃ for 4 min, followed by 30 cycles of 94℃ for 45 s, 55℃ for 45 s, and 72℃ for 2 min, and a final elongation step of 72℃ for 10 min. As a negative control, the template DNA was replaced by sterile double-distilled water.
For strain identification, the ITS sequences were compared with those in the NCBI database (http://www.ncbi.nlm.nih.gov/) using the BLAST search program for the final identification of the fungal endophytes program. A phylogenetic tree was constructed by the neighbor-joining method using MEGA version 5.
대상 데이터
The stability of the internal branches was assessed with 1,000 bootstrap replications. The sequences of this study were deposited in the GenBank database under the accession numbers: KP714353eKP714393 (Table 1).
이론/모형
notoginseng collected from Wenshan in August, 2012. The identification was performed by morphological and ITS sequencing methods. The pathogenicity was confirmed by experiments with detached roots of healthy notoginseng plants (unpublished data).
The crude extracts were stored at 4ºC before usage. The inhibitory effect of the EtOAc extracts obtained from endophytic fungi was tested using the double-layer agar technique [34] (Fig. 1D). The crude extracts were dissolved in 1.
The pure cultures were used for DNA extraction. DNA was extracted from 0.5-1.0 g mycelia chilled in liquid nitrogen by using the sodium dodecyl sulfate-cetyltrimethyl ammonium bromide method [31]. The internal transcribed spacer (ITS) region was amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') [32].
성능/효과
In this study, the diversity of endophytic fungi isolated from different tissues of healthy P. notoginseng was evaluated, and the isolates were screened for their potential antimicrobial activity against five major phytopathogens causing root rot of P. notoginseng. To the best of our knowledge, this is the first report of the biodiversity, phylogeny, and assessment of biocontrol potential of endophytic fungi harbored in P.
Among 89 endophytic fungi isolates,86 belonged to phylum Ascomycota, and three to Zygomycota. All isolates were classified to 12 orders (Capnodiales, Chaetothyriales, Diaporthales, Eurotiales, Glomerellales, Helotiales, Hypocreales, Microascales, Mucorales, Pleosporales, Sordariales, Xylariales), 23genera (Acremonium, Alternaria, Arthrinium, Aspergillus, Botryotinia, Chaetomium, Cladosporium, Colletotrichum, Dictyosporium, Fusarium, Humicola, Ilyonectria, Mucor, Myrothecium, Penicillium, Periconia, Pestalotiopsis, Phialophora, Phoma, Phomopsis, Plectosphaerella, Thielavia, and Trichoderma), in which isolate PH30448belonging to genus Arthrinium was incertae sedis at order level and isolate PH30402 was an unknown fungus belonging toSordariomycetes.
4%) exhibited activity against at least one of the pathogens tested. The highest frequency (16 isolates, 39.0%) was against the pathogen A. panax, followed by F. solani (15 isolates,36.6 %), F. oxysporum (14 isolates, 34.1%), Phoma herbarum (12 isolates, 29.3%), and M. acerina (7 isolates, 17.1%), respectively. Five isolates, PH30409, PH30412, PH30414, PH30441, and PH30444, were found to inhibit all pathogens tested, implicating they have a broad spectrum of antagonistic activity.
후속연구
acerina, and possibly other pathogens. Further studies are required to clarify the biocontrol active metabolites and the control efficiency of the potential endophytic isolates in both pot and field experiments. A full understanding of endophytic community structure, dynamics, and functions, and the shaping factors will be helpful in selecting pesticide, fertilizer, and other approaches for notoginseng planting.
참고문헌 (68)
1 He H.M. Cui L. Pharmacopoeia of the People's Republic of China 2010 Chinese Medical Science Press Beijing
2 Yang W.Z. Hu Y. Wu W.Y. Ye M. Guo D.A. Saponins in the genus Panax L. (Araliaeae): a systematic review of their chemical diversity Phytochemistry 106 2014 7 24 25108743
3 Zhang Y. Han L.F. Sakah K.J. Wu Z.Z. Liu L.L. Agyemang K. Bioactive protopanaxtriol saponins isolated from the roots of Panax notoginseng (Burk.) F. H. Chen Molecules 18 2013 10352 10366 24064450
4 Li X.T. Zhao Y.J. Jiang C.F. Zhang H.Q. Yu A.M. Determination of amino acids in Panax notoginseng by microwave hydrolysis and derivatization coupled with capillary zone electrophoresis detection Chem Res Chin Univ 29 2013 434 438
5 Jia D. Deng Y. Gao J. Liu X. Chu J. Shu Y. Neuroprotective effect of Panax notoginseng polysaccharides against focal cerebral ischemia reperfusion injury in rats Int J Biol Macromol 63 2014 177 180 24189392
6 Gao B. Huang L. Liu H. Wu H. Zhang E. Yang L. Wu X. Wang Z. Platelet P2Y 12 receptors are involved in the haemostatic effect of notoginsenoside Ft1, a saponin isolated from Panax notoginseng Brit J Pharmacol 171 2014 214 223 24117220
7 Yang Q. Wang X. Cui J. Wang P. Xiong M. Jia C. Liu L. Ning B. Li L. Wang W. Bidirectional regulation of angiogenesis and miR-18a expression by PNS in the mouse model of tumor complicated by myocardial ischemia BMC Complement Altern Med 14 2014 183 24903055
8 Zheng Y. Feng Z. You C. Jin Y. Hu X. Wang X. Han C. In vitro evaluation of Panax notoginseng Rg1 released from collagen/chitosan-gelatin microsphere scaffolds for angiogenesis BioMed Eng Online 12 2013 134 24380420
9 Zhou Q. Jiang L. Xu C. Luo D. Zeng C. Liu P. Yue M. Liu Y. Hu X. Hu H. Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis Thromb Res 133 2014 57 65 24196231
10 Meng X. Wang M. Wang X. Sun G. Ye J. Xu H. Sun X. Suppression of NADPH oxidase- and mitochondrion-derived superoxide by notoginsenoside R1 protects against cerebral ischemia–reperfusion injury through estrogen receptor-dependent activation of Akt/Nrf 2 pathways Free Radical Res 48 2014 823 838 24720662
11 Zhou N. Tang Y. Keep R.F. Ma X. Xiang J. Antioxidative effects of Panax notoginseng saponins in brain cells Phytomedicine 21 2014 1189 1195 24916704
12 Yan S. Li Z. Li H. Arancio O. Zhang W. Notoginsenoside R1 increases neuronal excitability and ameliorates synaptic and memory dysfunction following amyloid elevation Sci Rep 4 2014 6352 25213453
13 Zeng X.S. Zhou X.S. Luo F.C. Jia J.J. Qi L. Yang Z.X. Zhang W. Bai J. Comparative analysis of the neuroprotective effects of ginsenosides Rg1 and Rb1 extracted from Panax notoginseng against cerebral ischemia Can J Physiol Pharmacol 92 2014 102 108 24502632
14 Wang P. Cui J. Du X. Yang Q. Jia C. Xiong M. Yu X. Li L. Wang W. Chen Y. Panax notoginseng saponins (PNS) inhibits breast cancer metastasis J Ethnopharmacol 154 2014 663 671 24793216
15 Yang X. Xiong X. Wang H. Wang J. Protective effects of Panax notoginseng saponins on cardiovascular diseases: a comprehensive overview of experimental studies Evid Based Complement Alternat Med 2014 2014 204840 25152758
16 Gao D. Guo Y. Li X. Li X. Li Z. Xue M. Ou Z. Liu M. Yang M. Liu S. An aqueous extract of Radix astragali, Angelica sinensis , and Panax notoginseng is effective in preventing diabetic retinopathy Evid Based Complement Alternat Med 2013 2013 578165 23662142
17 Uzayisenga R. Ayeka P.A. Wang Y. Anti-diabetic potential of Panax notoginseng saponins (PNS): a review Phytother Res 28 2014 510 516 23846979
18 Yang X. Xiong X. Wang H. Yang G. Wang J. Xuesaitong soft capsule (Chinese patent medicine) for the treatment of unstable angina pectoris: a meta-analysis and systematic review Evid Based Complement Alternat Med 2013 2013 948319 24489594
19 Wang L. Li Z. Zhao X. Liu W. Liu Y. Yang J. Li X. Fan X. Cheng Y. A network study of Chinese medicine xuesaitong injection to elucidate a complex of action with multicompound, multitarget, and multipathway Evid Based Complement Alternat Med 2013 2013 652373 24058375
20 Ou X. Jin H. Guo L. Cui X. Xiao Y. Liu D. Huang L. Effects of balanced fertilization and soil amendment on growth and yield of Sanqi in continuous cropping China J Chinese Materia Medica 37 2012 1905 1911
21 Miao Z.Q. Li S.D. Liu X.Z. Chen Y.J. Li Y.H. Wang Y. Guo R.J. Xia Z.Y. Zhang K.Q. The causal microorganisms of Panax notoginseng root rot disease Scientia Agricultura Sinica 39 2006 1371 1378
22 Sun Y. Ke J. Ma N. Chen Z. Wang C. Cui X. Effects of root rot on saponin content in Panax notoginseng J Chin Med Mat 27 2004 79 80
23 Miao C.P. Qiao X.G. Zheng Y.K. Chen Y.W. Xu L.H. Guan H.L. Zhao L.X. First report of Fusarium flocciferum causing root rot of Sanqi ( Panax notoginseng ) in Yunnan, China Plant Disease 99 2015 1650
24 Hartley S.E. Eschen R. Horwood J.M. Gange A.C. Hill E.M. Infection by a foliar endophyte elicits novel arabidopside-based reactions in its host, Cirsium arvense New Phytologist 205 2015 816 827 25266631
25 Johnson L.J. de Bonth A.C.M. Briggs L.R. Caradus J.R. Finch S.C. Fleetwood D.J. Fletcher L.R. Hume D.E. Johnson R.D. Popay A.J. The exploitation of epichloae endophytes for agricultural benefit Fungal Divers 60 2013 171 188
26 Kusari P. Kusari S. Spiteller M. Kayser O. Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens Fungal Divers 60 2013 137 151
27 Mei C. Flinn B.S. The use of beneficial microbial endophytes for plant biomass and stress tolerance improvement Recent Pat Biotechnol 4 2010 81 95 20201804
28 Ma L. Cao Y.H. Cheng M.H. Huang Y. Mo M.H. Wang Y. Yang J.Z. Yang F.X. Phylogenetic diversity of bacterial endophytes of Panax notoginseng with antagonistic characteristics towards pathogens of root-rot disease complex Antonie van Leeuwenhoek 103 2013 299 312 22987248
29 Park S.U. Lim H.S. Park K.C. Park Y.H. Bae H. Fungal endophytes from three cultivars of Panax ginseng Meyer cultivated in Korea J Ginseng Res 36 2012 107 113 23717111
30 Qin S. Li J. Chen H.H. Zhao G.Z. Zhu W.Y. Jiang C.L. Xu L.H. Li W.J. Isolation, diversity, and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain forests in Xishuangbanna, China Appl Environ Microbiol 75 2009 6176 6186 19648362
31 Kim W.K. Mauthe W. Hausner G. Klassen G.R. Isolation of high molecular weight DNA and double-stranded RNAs from fungi Can J Bot 68 1990 1898 1902
32 White T.J. Bruns T. Lee S. Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics Protocols: a guide to methods and applications 1990 PCR Academic Press San Diego 315 322
33 Tamura K. Peterson D. Peterson N. Stecher G. Nei M. Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods Mol Biol Evol 28 2011 2731 2739 21546353
34 Santos S.B. Carvalho C.M. Sillankorva S. Nicolau A. Ferreira E.C. Azeredo J. The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique BMC Microbiol 9 2009 148 19627589
36 Zhang Q.H. Zhang J. Yang L. Zhang L. Jiang D.H. Chen W.D. Li G.Q. Diversity and biocontrol potential of endophytic fungi in Brassica napus Biol Control 72 2014 98 108
37 Hyde K.D. Soytong K. The fungal endophyte dilemma Fungal Divers 33 2008 163 173
38 Tejesvi M.V. Kajula M. Mattila S. Pirttilä A.M. Bioactivity and genetic diversity of endophytic fungi in Rhododendron tomentosum Harmaja Fungal Divers 47 2011 97 107
39 Eo J.K. Choi M.S. Eom A.H. Diversity of endophytic fungi isolated from Korea ginseng leaves Mycobiology 42 2014 147 151 25071383
40 Park Y.H. Kim Y.C. Park S.U. Lim H.S. Kim J.B. Cho B.K. Bae H. Age-dependent distribution of fungal endophytes in Panax ginseng roots cultivated in Korea J Ginseng Res 36 2012 327 333 23717135
41 Park Y.H. Lee S.G. Ahn D.J. Kwon T.R. Park S.U. Lim H.S. Bae H. Diversity of fungal endophytes in various tissues of Panax ginseng Meyer cultivated in Korea J Ginseng Res 36 2012 211 217 23717122
42 Wu H. Yang H.Y. You X.L. Li Y.H. Diversity of endophytic fungi from roots of Panax ginseng and their saponin yield capacities SpringerPlus 2 2013 107 23543782
43 Xing X. Guo S. Fu J. Biodiversity and distribution of endophytic fungi associated with Panax quinquefolium L. cultivated in a forest reserve Symbiosis 51 2010 161 166
44 Keswani C. Mishra S. Sarma B.K. Singh S.P. Singh H.B. Unraveling the efficient applications of secondary metabolites of various Trichoderma spp Appl Microbiol Biotechnol 98 2014 533 544 24276619
45 Böhm J. Hoff B. O’Gorman C.M. Wolfers S. Klix V. Binger D. Zadra I. Kürnsteiner H. Pöggeler S. Dyer P.S. Sexual reproduction and mating-type–mediated strain development in the penicillin-producing fungus Penicillium chrysogenum Proc Natl Acad Sci USA 110 2013 1476 1481 23307807
46 Jackson A.J. Walters D.R. Marshall G. Evaluation of Penicillium chrysogenum and its antifungal extracts as potential biological control agents against Botrytis fabae on faba beans Mycol Res 98 1994 1117 1126
47 Murali M. Sudisha J. Amruthesh K.N. Ito S.I. Shetty H.S. Rhizosphere fungus Penicillium chrysogenum promotes growth and induces defence-related genes and downy mildew disease resistance in pearl millet Plant Biology 15 2013 111 118 22672261
48 García M. Arriagada C. García-Romera I. Ocampo J.A. Are plant cell wall hydrolysing enzymes of saprobe fungi implicated in the biological control of the Verticillium dahlia pathogenesis? Crop Protect 30 2011 85 87
49 Liu K. Yang Y. Miao C.P. Zheng Y.K. Chen J.L. Chen Y.W. Xu L.H. Guan H.L. Ding Z.T. Zhao L.X. Koningiopisins A-H, polyketides with synergistic antifungal activities from the endophytic fungus Trichoderma koningiopsis Planta Medica 82 2016 371 376 26692458
50 Liu K. Yang Y.B. Chen J.L. Miao C.P. Wang Q. Zhou H. Chen Y.W. Li Y.Q. Ding Z.T. Zhao L.X. Koninginins N-Q, polyketides from the endophytic fungus Trichoderma koningiopsis harbored in Panax notoginseng Nat Prod Bioprospect 6 2016 49 55 26753920
51 Chen J.L. Sun S.Z. Miao C.P. Wu K. Chen Y.W. Xu L.H. Guan H.L. Zhao L.X. Endophytic Trichoderma gamsii YIM PH30019: a promising biocontrol agent with hyperosmolar, mycoparasitism, and antagonistic activities of induced volatile organic compounds on root-rot pathogenic fungi of Panax notoginseng J Ginseng Res 40 2016 315 324 27746683
52 Chen J.L. Liu K. Miao C.P. Sun S.Z. Chen Y.W. Xu L.H. Guan H.L. Zhao L.X. Salt tolerance of endophytic Trichoderma koningiopsis YIM PH30002 and its volatile organic compounds (VOCs) allelopathic activity against phytopathogens associated with Panax notoginseng Ann Microbiol 66 2016 981 990
53 Yang Y. Yang F. Miao C. Liu K. Li Q. Qin S. Zhao L. Ding Z. Antifungal metabolites from the rhizospheric Penicillium sp. YIM PH30003 associated with Panax notoginseng Phytochem Lett 11 2015 249 253
54 Li W. Yang X. Yang Y. Duang R. Chen G. Li X. Li Q. Qin S. Li S. Zhao L. Anti-phytopathogen, multi-target acetylcholinesterase inhibitory and antioxidant activities of metabolites from endophytic Chaetomium globosum Nat Prod Res 2016
55 Jin H. Yan Z. Liu Q. Yang X. Chen J. Qin B. Diversity and dynamics of fungal endophytes in leaves, stems and roots of Stellera chamaejasme L. in northwestern China Antonie van Leeuwenhoek 104 2013 949 963 23982430
56 Gazis R. Chaverri P. Diversity of fungal endophytes in leaves and stems of wild rubber trees ( Hevea brasiliensis ) in Peru Fungal Ecol 3 2010 240 254
58 Errasti A. Carmaran C. Novas V. Diversity and significance of fungal endophytes from living stems of naturalized trees from Argentina Fungal Divers 41 2010 29 40
59 Wearn J.A. Sutton B.C. Morley N.J. Gange A.C. Species and organ specificity of fungal endophytes in herbaceous grassland plants J Ecol 100 2012 1085 1092
60 Sánchez Márquez S. Bills G.F. Domínguez Acuña L. Zabalgogeazcoa I. Endophytic mycobiota of leaves and roots of the grass Holcus lanatus Fungal Divers 41 2010 115 123
61 Álvarez-Loayza P. White J.F. Jr Torres M.S. Balslev H. Kristiansen T. Svenning J.C. Gil N. Light converts endosymbiotic fungus to pathogen, influencing seedling survival and niche-space filling of a common tropical tree, Iriartea deltoidea PloS One 6 2011 e16386 21305008
62 Lana T.G. Azevedo J.L. Pomella A.W. Monteiro R.T. Silva C.B. Araújo W.L. Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis Gen Mol Res 10 2011 326 334
63 Eaton C.J. Cox M.P. Scott B. What triggers grass endophytes to switch from mutualism to pathogenism? Plant Sci 180 2011 190 195 21421360
65 Miao C.P. Mi Q.L. Qiao X.G. Zheng Y.K. Chen Y.W. Xu L.H. Guan H.L. Zhao L.X. Rhizospheric fungi of Panax notoginseng : diversity and antagonism to host phytopathogens J Ginseng Res 40 2016 127 134 27158233
66 Zhou S.L. Xiong G.M. Li Z.Y. Wen J. Loss of genetic diversity of domesticated Panax notoginseng F H Chen as evidenced by ITS Sequence and AFLP polymorphism: a comparative study with P. stipuleanatus H T Tsai et K M Feng J Integr Plant Biol 47 2005 107 115
67 Haas D. Défago G. Biological control of soil-borne pathogens by fluorescent pseudomonads Nat Rev Microbiol 3 2005 307 319 15759041
※ AI-Helper는 부적절한 답변을 할 수 있습니다.