两种八角莲内生真菌的生物活性研究

测定云南八角莲Dysosma aurantiocaulis和川八角莲Dysosma veitchii内生真菌的生物活性,为进一步从内生真菌中寻找活性成分提供指导,也为认识内生真菌相互作用提供有益的参考。从两种八角莲中分离出内生真菌187株,用滤纸片法测抗菌活性,用卤虫模型测抗卤虫活性得知大部分菌株发酵液都有活性,高活性菌株主要集中在镰孢霉属Fusarium、青霉属Penicillium、木霉属Trichoderma、丛梗孢属Monilia、毛壳菌属Chaetomium、无孢类群Mycellia sterilia、小齿梗霉属Rhinotrichum、无梗孢属Trichocladium和胶帚霉属Gliocladium。云南八角莲和川八角莲内生真菌是活性成分筛选的重要来源,并且通过活性分析发现:发酵液的活性强于菌丝体;云南八角莲的抗细菌活性菌株多于川八角莲。     

广西夹竹桃内生真菌的多样性和抑制几种水产病原菌活性筛选

为研究夹竹桃(Nerium oleander)内生真菌的多样性并评价其次生代谢产物的活性，该研究对广西夹竹桃(Nerium oleander)的内生真菌进行分离纯化，采用形态学和ITS序列分析结合的方法进行鉴定，以5种指示菌(其中有3种弧菌)对内生真菌提取物进行抑菌活性筛选。结果表明：(1)从广西夹竹桃中共得到19株内生真菌，这19株内生真菌都属于子囊菌门，涵盖5个目7个属，包括炭疽菌属(Colletotrichum)、球座菌属(Guignardia)、叶点霉属(Phyllosticta)、新壳梭孢属(Neofusicoccum)、曲霉属(Aspergillus)、隔孢壳科新属(Nothophoma)和间座壳属(Diaporthe),优势属为炭疽菌属(分离率为36.85%)和球座菌属(分离率为21.05%),其中炭疽菌属主要分布于茎，球座菌属全部来源于叶。(2)jing-117(Neofusicoccum sp.)和ye-130(Guignardia sp.)对坎氏弧菌有较为特异的抑菌效果，ye-136(Aspergillus sp.)能同时抑制蜡样芽孢杆菌和坎氏弧菌，ye-135(A...     

三种水生植物内生真菌多样性及其抗真菌活性

从华凤仙(Impatiens chinensis L.)、问荆(Equisetum arvense L.)和轮叶狐尾藻(Myriophyllum verticillatum L.) 3种植物中共分离到内生真菌155株。经鉴定, 它们分属于26个不同的分类单元。以杨桃炭疽菌(Colletotrichum gloeosporioides)、香蕉疫霉(Phytophthora nicotianae)和腐皮镰刀菌(Fusarium solani)等6种植物病原真菌为指示菌, 对3种水生植物内生真菌代谢产物进行抗菌活性研究。结果表明, 有37株内生真菌(占分离菌株总数的23.9%)显示出对一种或多种病原菌的抑菌活性。来自华凤仙、问荆和轮叶狐尾藻的抗菌活性菌株比例分别为29.2%、13.9%及37.1%, 都远低于已报道的陆生植物内生真菌中活性菌株的比例。抗菌活性菌株主要分布于枝孢属(Cladosporium)、木霉属(Trichoderma)和地霉属(Geotrichum)等5个属中。     

几种药用植物内生真菌抗真菌活性的初步研究

从三尖杉,南方红豆杉及香榧中分离出172株内生真菌,对其进行抗菌活性检测,结果表明共90株内生真菌对一种或多种植物病原真菌,如红色面孢霉（Neurospora sp.）,木霉(Trichoderma sp.）,镰刀菌（Fusarium sp.）等有抑制作用,来自三尖杉、南方红豆杉和香榧的抗菌活性菌株比例分别为40％,54.2%及57.1%。其中平板抑菌圈直径大于15mm的高抗菌株有35株。按Ainsworth等鉴定系统和方法进行鉴定,具有抗菌活性的内生真菌主要分布于心青霉属、镰孢菌属等18个属中。     

红树林内生真菌研究进展

红树林作为一种特殊的植物群落具有丰富的内生真菌资源,目前已分离鉴定的红树林真菌超过200种,成为海洋真菌的第二大类群,已报道的红树林内生真菌主要类群是链格孢霉(Alternaria)、曲霉(Aspergillus)、芽枝霉(Cladosporium)、炭疽菌(Colletotrichum)、镰孢霉(Fusarium)、拟青霉(Paecilomyces)、拟盘多毛孢(Pestalotiopsis)、青霉(Penicillium)、茎点霉(Phoma)、拟茎点霉(Phomopsis)、叶点霉(Phyllosticta)和木霉(Trichoderma)等.大部分红树林内生真菌具有较宽的宿主范围,极少数只有单一的宿主,不同红树林植物的内生真菌区系及优势种群有很大差异.红树林内生真菌的定殖因宿主植物不同部位、植株的年龄及季节和环境的变化明显不同.红树林内生真菌能产生多种代谢产物,具有抗菌、抗肿瘤等药用价值.红树林植物内生真菌的研究和开发具有重要意义.本文综述了红树林内生真菌的生物多样性及其分布、生物学功能和次生代谢产物等方面的研究进展.     

金荞麦和苦荞麦抗菌活性内生真菌的筛选及鉴定

从药用植物金荞麦和苦荞麦的根、茎、叶、花中分离到62株内生真菌,并以金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)[CMCC(B)63501]、小麦赤霉病菌(Fusarium graminearum)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cu-cumerinum)和绵腐病菌(Pythium aphanidermatum)6种微生物为指示菌对分离到的内生真菌进行抗菌活性检测。结果发现,分离的内生真菌菌株KQH-01、KQH-02和JQY-1的发酵醇提取物具有较好的抑菌活性。形态学特征和分子鉴定确定菌株KQH-01为炭角菌属(Xylaria sp.)真菌,菌株KQH-02为球毛壳菌(Chaetomium globosum),菌株JQY-1为葡萄座腔菌(Botryosphaeria dothidea)。     

除虫菊内生真菌类群与分布的初步研究

为了解除虫菊内生真菌的种类和分布情况,采用常规分离方法,从除虫菊(Pyrethrum cinerariifolium Trev.)根、茎、叶、花中分离获得内生真菌共计128株,经形态观察,鉴定出12个属:镰孢霉属(Fusarium),毛壳菌属(Chaetomium),茎点霉属(Phoma),链格孢属(Alternaria),内多隔孢属(Endophragmiella),拟盘多毛孢属(Pestalotiopsis),腐殖霉属(Humicola),黑孢霉属(Nigrospora),韦氏孢属(Wiesneriomyces),色串孢属(Torula),阜孢霉属(Papularia),丝核菌属(Rhizoctonia),其中镰孢菌属、链格孢属、腐殖霉属为优势种群。除虫菊不同部位内生真菌的数量、分布、种群及其组成存在差异。     

攀枝花苏铁植物内生真菌多样性的初步研究

为研究我国特有孓遗植物攀枝花苏铁(Cycas panzhihuaensis L.Zhou et S.Y.Yang)内生真菌的多样性,分别从原生境和迁移地采集不同部位的组织材料,经培养分离纯化菌株,并进行形态学鉴定和多样性分析.结果显示分离共获得内生真菌438株,其中346株分属3纲、5目、8科、37属,以交链孢属Alternaria sp.、盘多毛孢属Pestalotia sp.、镰孢霉属Fusarium sp.、黑孢霉属Nigrospora sp.为优势属,92株为无孢菌群,研究说明攀枝花苏铁内生真菌具有高度的生物多样性.研究还发现不同采集时间、不同生境多样性相似度较高,但不同组织部位的菌群组成差异比较明显.     

广西柳树桑寄生内生真菌的分离鉴定与抗肿瘤活性菌株筛选

该研究从广西钦州市采集健康的柳树桑寄生的根、茎和叶中分离并纯化内生真菌,对真菌进行形态学鉴定,提取内生真菌的DNA,采用真菌ITS序列对内生真菌进行分子鉴定。利用A549和H460细胞作为抗肿瘤活性指示细胞,采用MTT法测定真菌乙酸乙酯提取物的抗肿瘤活性。经过初步分离分析,从柳树桑寄生中纯化出27株内生真菌,经鉴定它们分别属于7个目9个属15个种。拟盘多毛孢属和间座壳属为优势属,其中拟盘多毛孢属全部定植于寄生根;其次为新壳梭孢属、拟茎点霉属和球座菌属,各分离到3株;其他包括青霉属、镰刀菌属、炭疽菌属和派伦霉属,各分离到1株。抗肿瘤活性研究表明,有一株与Pestalotiopsis protearum的ITS序列相似性达100%的拟盘多毛孢属菌株Gen24表现有抑制肿瘤细胞A549和H460生长的特性,在真菌乙酸乙酯浸提物浓度为800μg·mL~(-1)时,对A549细胞的抑制率达到了56.92%,对H460细胞的抑制率达到了70.11%。该研究结果表明广西柳树桑寄生内生真菌较丰富,在寄主中的分布表现了一定的组织特异性,而且还存在一些具有抗肿瘤活性的菌株及其活性物质可供进一步深入研究。     

南方红豆杉内生真菌的分离鉴定及其细胞毒活性研究

采用平板培养法对采自广东省的南方红豆杉进行内生真菌的分离,并通过形态学和分子生物学方法进行分类鉴定;共分离鉴定内生真菌22株,主要为刺盘孢属(Colletotrichum)、球座菌属(Guignardia)、座腔菌属(Botryosphaeria)、节菱孢属(Arthrinium)、炭角菌属(Xylaria)、毛壳菌属(Chaetomium)、交链孢属(Alternaria)、拟茎点霉属(Phomopsis)、长蠕孢属(Helminthosporium)等属。还采用MTT法测定了这些内生真菌发酵粗提物的细胞毒活性,活性研究发现有6个菌株的粗提物在作用浓度为100μg/mL时,对至少1种受试肿瘤细胞株的抑制率在50%以上,其中菌株A223和A240对人乳腺癌细胞MCF-7的抑制率在90%以上。表明南方红豆杉内生真菌多样性丰富,有些菌株具有良好的细胞毒活性,值得进一步深入研究。     

Nature and bioactivities of endolichenic fungi in Pseudocyphellaria sp., Parmotrema sp. and Usnea sp. at Hakgala montane forest in Sri Lanka

Aim:  The aim of this study was to investigate the nature and bioactivities of endolichenic fungi in three abundant lichens, Pseudocyphellaria sp., Usnea sp. and Parmotrema sp. in the lower elevation of Hakgala montane forest in Sri Lanka.
Methods and Results:  Endolichenic fungal strains, fungi that live asymptomatically in the lichen thallus, much the same way as endophytic fungi live within healthy plant tissues, were isolated from three abundant lichen species, Pseudocyphellaria sp., Usnea sp. and Parmotrema sp., at Hakgala montane forest in Sri Lanka, using the surface sterilization method. Nine endolichenic fungal strains were isolated from Parmotrema sp. and Usnea sp. separately, while 11 endolichenic fungi were recovered from the lichen Pseudocyphellaria sp. Isolation of endolichenic fungus Chrysosporium sp. 2 was common to all three lichen species. Substrate utilization patterns and antifungal activities of eight endolichenic fungal species were evaluated and the results revealed that all the test fungi were able to produce at least one enzyme to utilize the test substrates. Nigrospora sp., Chrysosporium sp. 1 and 2 and Cladosporium sp. showed antifungal activities on growth of some selected plant pathogenic fungi.
Conclusions:  Endolichenic fungal strains (29) were isolated from the lichens Parmotrema sp., Usnea sp. and Pseudocyphellaria sp. in Sri Lanka. Chrysosporium sp. 2 was common in all three lichens. Some of these endolichenic fungal strains showed antifungal activities against common plant pathogenic fungi and they are capable of utilizing the substrates by producing specific enzymes.
Significance and Impact of the Study:  The diversity and prevalence of the endolichenic fungi have not been studied extensively and this is the first report of isolation and identification of endolichenic fungi in lichens available in Sri Lanka.     

茅苍术内生真菌的分离鉴定及在组培苗中的回接

从茅苍术的叶、茎和根中分离鉴定内生真菌,并观测内生菌回接对茅苍术组培苗生长的影响。共获得茅苍术内生菌16株,其中叶片中14株,根茎中各获得一株。分别为交链孢霉、镰刀霉、小克银汉霉、青霉、茎点霉、小菌核菌、孔球孢霉和不产孢类。经内生菌和茅苍术快繁苗实验初筛,得到两株对植物无害的内生菌:小菌核菌和孔球孢霉,将它们回接到茅苍术组培苗中,可以从苗叶片中染色观察到菌丝。其中小菌核菌能提高茅苍术炼苗的存活率,接入内生菌的茅苍术叶片超氧化物歧化酶和过氧化物酶活性均高于未接菌的对照,脂肪酸不饱和指数基本不变,说明分离自茅苍术的内生菌回接后可以与宿主建立共生关系。     

蜘蛛抱蛋属植物内生细菌的多样性及其抗菌活性筛选

【背景】药用植物中蕴含多样性丰富的内生菌资源,这些微生物产生的多种新型物质在制药领域表现出较好的应用前景。【目的】研究蜘蛛抱蛋属(Aspidistra Ker-Gawl.)植物内生细菌的多样性,探索药用植物内生细菌在药用活性产物方面的开发潜力,以期发现具有抗菌活性的次级代谢产物。【方法】对9种13株新鲜的蜘蛛抱蛋植物进行表面消毒,采用5种分离培养基分离内生细菌;根据菌落形态特征排除重复菌株,并测定其16S rRNA基因序列,构建系统进化树分析内生细菌多样性;将菌株分别用2种培养基发酵,使用耻垢分枝杆菌(Mycobacterium smegmatis ATCC 700044)、水稻白叶枯菌(Xanthomonas oryzae PXO99A)、白色念珠菌(Candidaalbicans ATCC 10231)、肺炎雷伯菌(Klebsiella pneumoniae ATCC 700603)和耐药粪肠球菌HH22(Enterococcus faecalis HH22)5种检定菌对分离菌株的发酵液进行抑菌活性筛选。【结果】从植物组织中分离得到了234株内生细菌,根据形态初步排重得到156株植物内生细菌;基于16S rRNA基因序列构建的系统进化树显示它们分属于3门10目22科29属,其中链霉菌属(Streptomyces)、芽孢杆菌属(Bacillus)、微杆菌属(Microbacterium)、类芽孢杆菌属(Paenibacillus)和根瘤菌属(Rhizobium)的菌株广泛地分布在不同种的蜘蛛抱蛋植株中,且占据一定优势;发现可能的潜在新分类单元6个;156株内生细菌中38株菌的发酵液具有抑菌活性,初筛阳性率为23.7%。【结论】蜘蛛抱蛋植物组织中含有种类多样的内生细菌,它们可能是抗菌生物活性次级代谢产物的有效来源。     

Endophytic colonization ability of two deep-water rice endophytes, Pantoea sp. and Ochrobactrum sp. using green fluorescent protein reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized seeds of Jaisurya variety of deep-water rice viz., Pantoea sp. and Ochrobactrum sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp). Confocal laser scanning microscopy (CLSM) of hydroponically grown seedlings of Jaisurya rice, inoculated with gfp-tagged endophytes, revealed that both Pantoea sp. and Ochrobactrum sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp-tagged Ochrobactrum sp. was severely inhibited when co-inoculated with an equal number (10(5) c.f.u. ml(-1)) of wild type Pantoea sp., but the converse was not true. Pantoea sp. was a more aggressive endophytic colonizer of its host than Ochrobactrum sp. The potential of using GFP reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is herewith demonstrated.     

再接种内生真菌对葡萄叶内生真菌群落结构的影响

【目的】探讨回接内生真菌对葡萄叶内生真菌群落结构的影响。【方法】将葡萄叶中分离到的8株内生真菌回接到健康生长的玫瑰蜜葡萄叶片上,同时设置管理上喷施农药与未喷施农药两组处理,3个月后分析不同真菌菌剂处理后葡萄叶片内生真菌的分离率、优势度和多样性指数。【结果】5种菌剂的处理(Xylaria sp.、Nigrospora sp.、Alternaria sp.1、Alternaria sp.2和Colletotrichum sp.)在处理后的叶片中分离到了较多数量的接种菌。不同菌剂处理后葡萄叶片菌群结构差异显著。【结论】与对照处理的葡萄相比,菌剂的处理增加了叶内生真菌的分离率,但显著降低了内生真菌的多样性指数;发现喷施农药组叶片内生真菌的分离率较低,但多样性相对较高。     

柑橘黄龙病寄主长春花内生细菌的分离及功能鉴定

【目的】感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构及功能对柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】利用兼性厌氧可培养技术以及植物内生菌功能特性分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。29种内生细菌进行功能分析,其中6株内生细菌至少具有4种功能特性,分属于马胃葡萄球菌、苏云金芽孢杆菌、巨大芽孢杆菌、短小杆菌属、摩氏摩根菌(Morganella morganii)及溶杆菌属(Lysobacter sp.)5个属。【结论】感病与健康长春花植株中均含有丰富的内生细菌且差异较大,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构。通过分析菌群的差异,有望找到与柑橘黄龙病菌生长相关的菌种。     

Antitumor and antifungal activities in endophytic fungi isolated from pharmaceutical plants Taxus mairei, Cephalataxus fortunei and Torreya grandis

The purpose of this work was to screen the endophytic fungi having antitumor or antifungal activity, which were isolated from the inner barks of three kinds of pharmaceutical plants, Taxus mairei, Cephalataxus fortunei and Torreya grandis, collected from Fujian province, China. Antitumor activity was studied by the MTT assay and antifungal activity was determined by observing fungal growth inhibition. 13.4% of endophytic fungi fermentation broths displayed cytotoxic activity on HL-60 cells at and below a dilution of 1:50, and 6.4% on KB cells. 52.3% of endophytic fungi fermentation broths displayed growth inhibition on at least one pathogenic fungi, such as Neurospora sp., Trichoderma sp. and Fusarium sp. Among all endophytic fungi isolated, the genus Paecilomyces sp. has the highest positive rate of antitumor and antifungal activity. These results indicate that endophytic fungi could be a promising source for antitumor and antifungal bioactive agents.     

Study of endophytic fungal community from different parts of <Emphasis Type="Italic">Aegle marmelos</Emphasis> Correae (Rutaceae) from Varanasi (India)

Endophytic fungi were isolated from healthy, living, and symptomless tissues of inner bark, leaf, and roots of Aegle marmelos, a well-known medicinal plant, growing in different parts of India including Varanasi. A total of 79 isolates of endophytic fungi were isolated, representing 21 genera, adopting a standard isolation protocol. Members of the deuteromycotina were more prevalent than ascomycotina and others. The result was quite encouraging in terms of maximum isolates recovery from hyphomycetes (78.5%) followed by ascomycetes (8.9%) and coelomycetes (7.6%) respectively, which corroborates previous studies in same area. However, 5.1% isolates remained unidentified and were classified under Mycelia Sterilia. No isolate was obtained from either basidiomycotina or from zygomycotina. Fusarium spp. had maximum colonization frequency (8.00%) in this plant. The other dominant endophytic genera were Aspergillus spp., Alternaria sp., Drechslera sp., Rhizoctonia sp., Curvularia sp., Nigrospora sp., and Stenella sp. Only two ascomycetous members Chaetomium globosum and Emericella sp. (perfect state of Aspergillus sp.) were obtained from the bark sample. These results indicated that distribution of endophytic fungi within the A. marmelos is not even. Bark harbors more endophytic fungi than leaf and root.     

柑橘黄龙病赣南脐橙内生菌种群结构分析

【目的】分析赣南脐橙黄龙病植株和健康植株叶片内生菌,对比不同培养条件下培养出的内生菌,为筛选出对柑橘黄龙病原菌有影响的伴生菌奠定理论基础。【方法】通过PCR方法对脐橙中黄龙病菌进行验证,并基于16S r RNA基因高通量测序技术对患病与健康赣南脐橙叶片内生菌以及不同培养基富集培养后的内生菌进行多样性分析。【结果】所采集样品中有5株患病株,5株健康株。5株病株中共同含有的细菌属有13个,其中7个在5株健株中也共同存在。Defluviicoccus属和Granulicella属在病健株植物中都是优势菌属,且在健株中的平均含量高于病株。病株与健株的样品相似度存在明显界线。富集培养后不同样本和不同培养基中菌属分布不同。肠杆菌属(Enterobacter)、短小杆菌属(Curtobacterium)、假单胞菌属(Pseudomonas)和泛菌属(Pantoea)得到了大量富集,不动杆菌属和沙雷氏菌属等9个菌属富集量较少。另外,培养和未培养各样本间未分类菌(Unclassified)含量差异也较大。【结论】赣南脐橙患病植株和健康植株叶片内生菌有着明显差异,黄龙病菌的存在改变了脐橙叶片原有内生细菌的菌群结构。从活体植物组织内直接检测才能得到真正的植物内生菌群落分布情况。通过分析菌群的差异,有望找到与柑橘黄龙病菌生长相关的伴生菌。     

回接内生真菌对工业大麻生理及农艺性状的影响

回接工业大麻中分离出的6种内生真菌于大田种植的云麻1号,研究了内生真菌对工业大麻酶活性及农艺性状的影响。结果表明：接种毛壳菌和镰孢菌的大麻植株SOD和POD活性显著提高（P<005）;除炭疽菌外,小不整球壳菌、毛壳菌、黑孢霉、镰孢菌和粘束孢均提高了大麻植株的麻皮干重和麻皮厚度;毛壳菌处理组大麻植株的节间长、麻皮干重、麻皮厚度均显著高于对照（P<005）。这些结果为大麻生产中合理利用内生真菌制剂促进大麻优质丰产及深入研究内生真菌的作用机制奠定基础。