【作者】 王增福；

【导师】 钱晓鸣；

【作者基本信息】 厦门大学 ， 微生物学， 2008， 硕士

【摘要】 20世纪90年代以后,植物内生真菌研究范围扩大,不再仅限于禾本科植物,开始倾向从木本植物体内分离内生真菌,研究其生物多样性、生物学作用、内生机理、与宿主之间的关系以及新型活性化合物研究等。本文对一个引种实验葡萄园中酿酒葡萄的内生真菌资源进行调查,分析其群落构成和种群系统发育关系,对这些菌株的抗植物病原真菌、抗人类病原菌活性进行了初步研究;对活性菌株A23的代谢产物进行活性追踪,对其分离纯化得到的化合物进行结构鉴定和抗植物病原真菌活性研究;同时对同一葡萄园的果实所酿造的葡萄酒进行检测,确定分离到的内生真菌代谢产物Enniatin B是否对葡萄酒造成污染。从酿酒葡萄植株的根、枝条、叶片、果实等组织共分离得到内生真菌455株,统计结果显示,内生真菌的种类和数量与宿主健康状况相关,引种成活率较高的植株内生真菌分离率较高,而引种成活率低的植株内生真菌分离率相应很低,分析其群落构成和发育关系,表明Alternaria,Penicillium,Fusarium是优势种群,一些真菌为潜在的植物病原真菌,如Diapor the phaseolorum,Aspergillusoryzae,Fusarium equiseti,Pestalotiopsis uvicola等;以黄瓜炭疽病菌(Colletotrichum Jagenarium(Passerini))、棉花黄萎病菌(Verticilliumdahliae Kleb)、棉疫霉果腐病菌(Phytophthora parasitica Destur)为指示菌,初筛采用对峙法测定了103株内生真菌的抗菌活性,复筛使用滤纸片法,初筛得到活性菌株30株,占总数的29.1%,复筛显示,有8株菌对短小芽孢杆菌和枯草芽孢杆菌的抑制作用超过氨苄青霉素,并且其中13株对3种植物病原指示菌均有抗性,显示了一定的广谱抗性,在这13株菌中,10株属于优势种群。表明酿酒葡萄蕴含丰富的内生真菌资源,优势种群经过种间竞争,通过产生多种活性代谢产物增加宿主的抗病害、抗逆能力。在研究酿酒葡萄内生真菌发育关系时发现一株内生真菌属于虫草属真菌,与文献中报道的昆虫内生真菌Cordyceps建立系统发育树,表明植物内生真菌Cordyceps与昆虫内生Cordyceps真菌有较大差异,分别形成独立的类群,在其发育史上可能通过基因调节经历了从植物到昆虫的宿主“跳跃”。菌株A23经过分子鉴定为Fusarium tricincrum(Corda),本文采用固体培养、有机溶剂提取、分相萃取、柱层析等分离纯化方法从其发酵产物中分离到5个纯的化合物,经过核磁共振等波谱解析确定其中两个化合物的结构相同,为Enniatin B。测定其抗植物病原真菌生长活性,发现Enniatin B有广谱抗性,且活性较好,对黄瓜炭疽病菌MIC为7μg/mL,对棉花黄萎病菌MIC为3μg/mL,对棉疫霉果腐病菌MIC为15μg/mL,显示Enniatin B具有开发成为农用抗生素的潜力。为检测葡萄酒是否被对人类有害的内生真菌代谢产物污染,对采用同一葡萄园收获果实酿造的两种葡萄酒(红葡萄酒和白葡萄酒)进行高压液相分析,结果显示红葡萄酒内不含Enniatin B,白葡萄酒内则含有Enniatin B,浓度达到11.04μg/mL。表明植物内生真菌及其代谢物有可能通过一定途径进入植物产品,引发食品安全问题。本论文研究表明,酿酒葡萄蕴含丰富的内生真菌资源,是研究内生真菌-宿主发育关系的良好工具,同时蕴含着丰富的活性物质,是寻找新型农药和人用抗生素的有效来源;但也可能会产生对人类有害的毒素,并进入人类食品,引发其安全问题,表明植物内生真菌一方面可以提高宿主的抗逆性,为人类提供有益的活性化合物,对人类农、林、牧业生产活动产生积极影响,另一方面植物内生真菌也可能降低农、林、牧业产值、威胁食品安全,对人类生活有双重作用。更多还原

【Abstract】 Since 1990s,the range of plant endophytic fungi study has been expanded,not being limited gramineous plants only,but tending to isolate endophytic fungi from woody plants to explore the biological diversity,biology influence,endophytic mechanism,relation with host,and the exploration of new bioactive compounds,etc.This thesis focused on the endophytic fungi of Vitis vinifera in an introduction experimental vineyard,analyzed the community constituents and the phylogenetic relation,supplied a preliminary study on the inhibitory activity of the strains to phytopathogenic and human pathogen,investigated the constituents and the inhibitory activity to phytopathogenic fungi of the compounds which were traced and isolated from the metabolites of active strain A23,and inspected whether the wine brewed from the fruits in this vineyard was polluted or not by the endophytic fungi’s metabolites harmful to human beings.455 endophytic fungi strains were isolated from the roots,branches, leaves,and fruits of Vitis viniferas.The statistics indicated that the types and quantities of the endophytic fungi were related with host’s health situation.The separation rate of the endophytic fungi from plants with high survival ratio was relevantly high,and the vice versa.The analysis of community constituents and phylogenetic relation demonstrated that Alternaria,Penicillium,Fusarium were dominant species,but among which there were some potential phytopathogenic fungi,such as Diaporthe phaseolorum,Aspergillus oryzae, Fusarium equiseti,Pestalotiopsis uvicola,etc.Taked Colletotrichum lagenarium(Passerini),Verticillium dahliae Kleb,and Phytophthora parasitica Destur as indicator fungi,the primary screening employed antagonistic method to check the antiseptic activity of 103 endophytic fungi,while rescreening used filter paper method.Thirty active strains were obtained from the primary screening,accounting for 29.1%of the total strains.The rescreening displayed that there were eight strains whose inhibition to Bacillus pumilus and Bacillus subtilis surpassed ampicillin,and thirteen strains,among which seven strains belonged to dominant species,had resistance to the three above-mentioned indicator fungi,which indicated a certain broad spectrum resistance.The results demonstrated that Vitis vinifera consisted of abundant endophytic fungi. Through the interspecific competition,the dominant species created various active metabolites to improve the host’s anti-disease and anti-adversity ability, which proved the view of mutual choice and mutual adaptation between host and endophytic fungi,and "The Host Relatedness Hypothesis".One endophytic fungus was found out to belong to Cordyceps fungi during the research of endophytic fungi phylogenetic relation,and the phylogenetic tree was created together with insect Cordyceps fungi reported in papers.The result indicated that the Cordyceps fungi in plants were different with those in insects;they had become independent species;and a "leap" from plant to insects’ host had happened through the gene regulation during the developmental process.The Vitis vinifera belonged to one of the segments.The strain A23 belonged to Fusarium tricinctum(Corda) through the molecular identification.The separation methods,such as solid culture, extraction,Silica Column Chromatography,Dextran gel Column Chromatography(HPLC),etc.were employed in this thesis to get five pure compounds from the fermentation production.The spectral analysis,such as NMR,displayed that the structure of two compounds was the same,that was, Enniatin B.Its inhibitory activity of phytopathogen indicated that Enniatin B had broad spectrum resistance and good activity:the MIC for Colletotrichum lagenarium was 7μg/mL,for Verticillium dahliae,the MIC was 3μg/mL,and for Phytophthora parasitica MIC was 15μg/mL.Combined with pot experiment test,Enniatin B was proved to have the potentiality to be qualified agriculture antibiotics.To check whether the wine was polluted by the endophytic fungi’s metabolites that was harmful to human beings,two kinds of wine(red wine and white wine) brewed from the fruits in the same vineyard were analyzed by HPLC.The result proved that red wine dides not comprise Enniatin B,while white wine comprises it,reaching to 11.04μg/mL.It illustrated that the plant endophytic fungi and their metabolites may entered into plant production through some certain ways,which may generated food safety problems.The present study proved that with abundant endophytic fungi,Vitis vinifera was a good way to investigate endophytic fungi and host phylogenetic relation.And with large quantity of active compounds,it was also an effective way to find out new agricultural chemicals and human antibiotics.However, harmful toxinum may be producted and taken into human food,generating food safety problems.On the one hand,plant endophytic fungi can improve host’s anti-adversity ability,supply useful active compounds for human beings, and make a contribution to agriculture,forestry and livestock production,on the other hand,they may reduce the output value of agriculture,forestry and livestock,and threaten food safety.They have the double-response to human life.更多还原