| Objective: To study the species,dominant species and genus of Dendrobium huoshanense endophytic fungi under different cultivation conditions,as well as which were distributed in the different tissue.To screen out the dominant species / genus of antipathogenic fungi.Mealwhile,those which were explored by its secondary metabolites,hoping that there are obtained the same or the similar active compounds with the host plant,especially including novel structure and the higher biological activity of the pilot compounds.Ultimatly,it will lay the scientific basis that is conducive to achieve the sustainable development of resources and quality control of authentic herbs Dendrobium huoshanense in the near future.Methods: The endophytic fungi of Dendrobium huoshanense in different cultivation environments were isloted and identified by the technology of classical tissue block separation and molecular biology.Moveover,there were analyzed that was based on the principles of biostatistics,including diversity and community structure.The dominant strains that contain activity of antipathogenic fungal were not only screened by the crossroads,but also were large-scale fermented.The fermentation broth was separated and identified by various chromatographic methods and modern spectroscopic techniques.Results: 168 endophytic fungi were isolated and identified from Dendrobium huoshanense in 7 different cultivated environments,belonging to 18 genera and 36 species.The Margaef index,the Pielou index and the Shannon index in-dicated that the endophytic fungi of shennong that cultivated on wood chipsis were more abundant than other environments.In the terms of the plant tissue,the type and quantity of endophytic fungi in the stems were more than the ro-ots and leaves.Stagonosporopsis oculihominis,Alternaria eichhorniae,Phyllosticta aristolochiicola,Aspergillus flavus,Fusarium lactis,there were five strains which were dominant strains.The extract of Aspergillus flavus had the bad antipathogenic fungi.effect against 10 pathogenic fungi.The other four kinds of extract against the 10 kinds of pathogens have their own advantages and disadvantages,in which the average inhibition rate of Stagonosporopsis oculihominis to 10 pathogenic fungi was m-ore than 50%,especially for Sclerotinia sclerotiorum inhibition rate of up to 90%.Compared to other strains aganist the three kinds of Dendrobium pathogens,the extract of Alternaria eichhorniae has a broad spectrum of antipathogenic fungi effect.Rhizoctonia solani can be inhibited to 100% by using the extract of Fusarium lactis.Isolated and identified a total 74 compounds from Alternaria eichhorniae,Stagonosporopsis oculihominis and Fusarium lactis,those which showed mode-rate biological activity.A number of 36 compounds were isolated and identified in the Stagonosporopsis oculihominis,as follows: stagonoculiepine(1),stagonoculiazin(2),acetyl-L-isoleucine(3),N-(4-oxopentyl)acetamide(4),(E)-4-(4-hydroxyphenyl)but-3-en-2-one(5),p-hydroxyphenylethanol(6),p-hydroxyphenylacetic acid(7),Nb-(2-(1H-indol-3-yl)ethyl)acetamide(8),N-Isobutylacetamide(9),ethyl-succinate(10),Cyclo-(D)-alanine-(L)-proline(11),uracil(12),Cyclo-(L)-proline-(L)-phenylalanine(13),Cyclo-(D)-proline-(L)-phenylalanine(14),1H-indole-3-carboxylic acid(15),2-(1H-indol-3-yl)acetic acid(16),adenine nucleoside(17),5-methoxydaidzein(18),Cyclo-(L)-proline-(L)-valine(19),Cyclo-(L)-proline-(D)-valine(20),Cyclo-(L)-proline-(L)-isoleucine(21),Cyclo-(L)-proline-(D)-leucine(22),Cyclo-(L)-proline-(L)-leucine(23),Cyclo [D-(4-hydroxyprolinyl)]-(L)-leucine(24),Cyclo[L-(4-hydroxyprolinyl)]-(L)-leucine(25),Cyclo-(L)-proline-(L)-proline(26),Cyclo-(L)-tyrosine-(L)-proline(27),Cyclo-(L)-phenylalanine-(L)-leucine(28),(Cyclo-(L)-4-OH-proline-(L)-phenylalanine(29),oleic acid(30),?-linoleic acid(31),8(E)-hexadecenoic acid(32),linolenic acid(33),?-linoleate(34),22E-7?methoxy-5?,6?-epoxyergosta-8(14),22-dien-3?-ol(35),demethylincisterol(36).A number of 17 compounds were isolated and identified in the Alternaria eichhorniae,as follows: p-hydroxyphenylacetic acid(37),(2S,3R)-1-phenylbutane-2,3-diol(38),p-hydroxyphenylethanol(39),3-hydroxy-4-(4-hydroxyphenyl)-2-butanone(40),(2R,3S)-1-(4-hydroxyphenyl)butane-2,3-diol(41),tricycloalternarenes 6b(42),tricycloalternarenes 1b(43),tricycloalternarenes H(44),tricycloalternarenes 1a(45),tricycloalternarenes 2a(46),Nb-(2-(1H-indol-3-yl)ethyl)acetamide(47),Cyclo-proline-phenylalanine(48),altertoxins I(49),stemphyperylenol(50),alterperylenol(51),melithasterol B(52),(22E,24R)-3?,5?,9?-Trihydroxyergosta-7,22-dien-6-one(53).A number of 21 compounds were isolated and identified in the Fusarium lactis,as follows: N-phenethylacetamide(54),1H-indole-3-carbaldehyde(55),thymidine(56),uracil(57),lignoren(58),uridine(59),hexahydrate(60),adenosine(61),Cyclo-glycine-(L)-proline(62),Cyclo(D)-proline-(L)-phenylalanine(63),Cyclo(L)-proline-(L)-phenylalanine(64),2-pyrrolidinone(65),N-methyl-2-pyrolidinone(66),Cyclo-(L)-4-OH-proline-(L)-phenylalanine(67),brevianamide F(68),3-methyl-piperazine-2,5-dione(69),7,8-dimethylbenzo[g]pteridine-2,4(1H,3H)-dione(70),Cyclo(L)-tyrosine-(L)-phenylalanine(71),beer sterols(72),daidzein(73),erythritol(74).Conclusion: The community structure of Dendrobium huoshanense endophytic fungus under different cultivation environments was rich,However,there were not only different species for endophytic fungus that in the different cultivation environments,but also were diversity in different tissue with host plant.The dominant species of the endophytic fungus had broad spectrum of antip-athogenic fungi effect,especially for the pathogen of Dendrobium huoshanense.74 compounds were isolated,including 64 different compounds,compound 1-2 was the new compound and compound 3 was a new natural product.The above compounds were isolated from three endophytic fungus first time. |
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