资源植物水柏枝属的分类与应用研究进展

水柏枝属是一类具有重要生态、经济意义的资源植物,该文対国内外关于水柏枝属分类学、系统学、内生菌、繁育和保护以及药用成分等方面的研究进展进行综述。并针对该属存在的有关物种界定与谱系地理、植物内生菌与适应性机理、药用成分与药理研究等关键科学问题,提出可能采用的研究方法,为高效利用野生资源植物提供基本思路。     

植物内生菌研究进展

植物内生菌是指生活史中某一阶段生活在植物组织内,对植物没有明显病害症状的一类微生物,研究发现其主要包括内生细菌、内生真菌和内生放线菌。内生菌能够产生与宿主植物相同或相似的活性物质,从而拓宽了药用资源并且具有巨大的应用价值。从植物内生菌的分布规律及其生长特点,与宿主植物的关系,研究领域以及发展现状与存在问题等方面进行了综述,并对植物内生菌在药用资源开发中的前景进行了展望。     

中国水柏枝属植物的地理分布、濒危状况及其保育策略

水柏枝属(MyricariaDesv.)植物主要分布于中国青藏高原及其邻近地区,为欧亚温带高山植物类群,多为河岸带植物,是分布区湿地生态系统和荒漠生态系统的重要组分。为评价这些特殊生态系统关键植物的分布及受威胁现状,对中国水柏枝属植物的地理分布和濒危状况进行了调查。调查发现,该属中国分布的11个分类群均受到生存胁迫,其中疏花水柏枝即将成为野外灭绝种,宽叶水柏枝已属极危种,心叶水柏枝为濒危种,秀丽水柏枝、泽当水柏枝、小花水柏枝、卧生水柏枝和匍匐水柏枝为渐危种,具鳞水柏枝、三春水柏枝和宽苞水柏枝为受胁种。结果表明,以水利建设为主的人为干扰和土地沙化、气温升高带来的环境改变是导致该属植物生存胁迫的主要原因。最后探讨了水柏枝属这类河岸带湿地和荒漠生境植物的保育策略。     

乌头属植物化学成分、药理作用及其内生菌的研究进展

毛茛科乌头属植物广泛分布于北温带地区,具有重要的药用价值。乌头属植物主要的化学成分是生物碱,其中二萜生物碱是历来的研究热点。现代药理学研究证明,乌头属植物具有镇痛、抗炎、抗肿瘤及杀虫等作用。目前乌头属植物内生菌的研究也逐渐开展。本文对乌头属植物近年来报道的新的化学成分、药理研究和利用植物内生菌产生活性成分等进行了综述,可为乌头属植物的进一步开发利用提供参考。     

珍稀药用石斛内生菌研究进展

药用石斛是我国传统名贵中药材,具有重要的药用价值,由于人为和环境等因素,药用石斛资源已变得极其匮乏,难以满足市场的需求。药用石斛内生菌资源丰富,在与宿主石斛长期共生的过程中,对药用石斛的生长发育、环境的适应、活性代谢产物的积累等方面具有很强的促进作用。深入研究药用石斛内生菌及其与宿主的关系,对药用石斛生产、资源保护与利用有着重要意义。本文简要概述了药用石斛内生菌资源及其生物学功能,并探讨了存在的问题和未来发展方向,以期为药用石斛内生菌的开发与利用提供参考。     

植物内生菌活性代谢产物最新研究进展

内生菌广泛存在于植物组织中,作为一种新型的微生物资源,具有丰富的物种多样性,也是发掘新型天然活性物质的重要途径之一,有些内生菌还能产生与宿主植物相同或相似的活性成分。因此从植物内生菌中挖掘抗菌尤其抗临床耐药菌、抗肿瘤等天然活性产物不仅为新药的研发提供了新的方向,还能在一定程度上解决传统的天然产物药源——药用植物生长缓慢、资源紧缺等问题。从多个角度概述了近年来国内外报道的植物内生菌次生代谢产物及其来源、生物活性等方面的主要成果和最新进展,以期为植物内生菌活性代谢产物的研究提供参考。     

中国种子植物内生真菌资源及菌植协同进化

综述了中国种子植物内生真菌资源研究概况,比较了裸子植物和被子植物内生真菌种类,它们都具有肉座菌目(Hypocreales),粪壳菌目(Sordariales),散囊菌目(Eurotiales),毛霉目(Mucorales)及不产孢类(Myceliasterilia)内生真菌。裸子植物内生真菌涉及52个属,既包括高等的子囊菌和担子菌,也包括低等的卵菌(Oomycetes)和接合菌(Zygomycetes)类。被子植物涉及60个属,主要为高等的子囊菌(Ascomycetes)和担子菌(Basidiomycetes),低等的卵菌和接合菌报道很少。双子叶植物涉及40个属,单子叶植物内生真菌涉及30个属,两类被子植物所报道的内生真菌只有11个属相同。裸子植物与双子叶植物内生真菌相似程度较高,都具有炭角菌目(Xylariales)、格孢腔菌目(Pleosporales)、柔膜菌目(Helotiales)和白粉菌目(Erysiphales),刺盘孢菌属(Colletotrichum)、拟茎点霉属(Phomopsis)、枝孢霉属(Cladosporium)、地霉属(Geotrichum)等内真菌,共20个属相同。各类种子植物具有自己独特的一些内生真菌。还对植物与其内生真菌的协同进化关系进行了分析。     

药用植物的结构、发育与其主要药用成分积累关系的研究

药用植物防病治病的基础是含有药用化学成分,这些药用成分都贮存在它们的药用部分(根、茎、树皮、叶、花、果实或种子等)内.为此,研究药用植物的结构、发育与其主要药用成分积累的关系,具有重要的理论和实践意义.应用植物解剖学、组织化学和植物化学等技术相结合的方法,先后研究和阐明了西洋参、金丝桃属和芦荟属植物、绞股蓝、盾叶薯蓣、青藤和毛青藤以及地黄等药用植物的主要药用化学成分在药用部分中积累的部位、贮存结构的特点,在植物不同生长发育时期中药用成分含量的变化规律以及同属不同种植物的结构与药用成分积累的关系.研究结果为药用植物的合理采收、制定植物药材的SOP、GAP,以及药用植物资源的开发利用和保护提供了科学依据.     

药用植物黄花蒿内生菌的研究进展

黄花蒿作为治疗疟疾最有效的中草药植物,同时还具有其他多种药用和农业生产价值。与其密切相关的内生菌资源已是目前研究的一大热点。总结整理了黄花蒿内生菌的研究现状,其应用主要集中在抗肿瘤抗氧化活性、抑菌活性、促进青蒿素的合成等方面。     

药用石斛内生菌研究进展

综述了药用石斛内生菌的分离鉴定以及与宿主互作关系等方面的国内外研究进展。在介绍药用石斛内生真菌、内生细菌的基础上，结合本课题组前期对石斛内生细菌做出的相关研究工作，对其研究发展思路进行了展望，为开发安全有效的微生物菌肥、中医药产品、新资源功能性食品，合理利用药用石斛资源提供科学依据。     

Endophytic Fungal Assemblages from Inner Bark and Twig of Terminalia arjuna W. & A. (Combretaceae)

Summary Fungal endophytes reside in healthy tissues of all terrestrial plant taxa studied to date and are diverse and abundant in tropical woody angiosperms. Endophytic fungi were isolated from Terminalia arjuna, an important ethno pharmacological plant extensively used in ayurvedic medicines to treat heart ailments. Isolations were made from symptomless fresh inner bark as well as twig samples of five plants collected from three locations of riparian vegetation during two seasons (monsoon and winter) of 2003 and 2004. Two hundred and seventy eight isolates, representing 22 genera, were obtained from both seasons. Monsoon seasonal isolations representing 22 genera showed greater diversity. Coelomycetes were more numerous during the winter season than hyphomycetes and ascomycetes. Among the endophytes, the genus Pestalotiopsis dominated the endophyte assemblage of T. arjuna collected from different locations, dominance was greater during the winter season than the monsoon season. Endophytic colonization frequency was greater in inner bark (18.5%) than twigs (4.6%). The genera Pestalotiopsis (54.5%), Chaetomium (10.5%) and Myrothecium (9%) were the most predominant endophytes. Rarefaction indices indicated the highest expected number of species for bark samples, monsoon isolations and location 1 (Mysore).     

基于高通量测序分析西藏沙棘根瘤内生菌的多样性

根瘤是微生物侵染植物根部并与之形成的共生结构,这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌,而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌,这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物,由于其分布生境的特殊性,对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究,培养方法因难以模拟自然条件而不易获得纯培养,高通量测序技术对其多样性的研究提供了便利。因此,本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料,采用16S rRNA基因扩增子高通量测序方法,结合OTU分析,对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明,西藏沙棘根瘤内生菌具有丰富的多样性,根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia）,其相对丰度为47.63%,共检测到7个弗兰克氏菌属的OTUs;根瘤内除弗兰克氏菌外,还存在大量的非弗兰克氏菌,共检测到1523个OTUs,隶属于22个门、33个纲、69个目、113个科和202个属,相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明,西藏沙棘根瘤内生菌具有丰富的多样性,西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌,并且还分布着非弗兰克氏菌;在同一根瘤样品中,弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法,还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。     

Fungal endophytes in seeds and needles of Pinus monticola

Using a sequence-based approach, we investigated the transmission of diverse fungal endophytes in seed and needles of Pinus monticola, western white pine. We isolated 2003 fungal endophytes from 750 surface-sterilized needles. In contrast, only 16 endophytic isolates were obtained from 800 surface-sterilized seeds. The ITS region was sequenced from a representative selection of these endophytes. Isolates were then assigned to the most closely related taxa in GenBank. Although 95 % of the endophytes in needles from mature trees belonged to the Rhytismataceae, 82 unique ITS sequences were obtained from at least 21 genera and 10 different orders of fungi. Significantly, none of the endophytes in seed were rhytismataceous (χ² = 180; P < 0.001). Similarly, needles of greenhouse seedlings yielded only non-rhytismataceous isolates, whereas seedlings of the same age that had naturally regenerated near older white pines in roadless areas were colonized by rhytismataceous endophytes almost to the same extent as in mature trees. Only one of 17 rhytismataceous isolates were able to grow on a medium containing only 0.17 % nitrogen, whereas 25 of 31 non-rhytismataceous endophytes grew. Rhytismataceous endophytes are dominant in needles of P. monticola, but they appear to be absent in seed, and unlikely colonists of nitrogen-limiting host tissues such as the apoplast.     

General microflora,arbuscular mycorrhizal colonization and occurrence of endophytes in the rhizosphere of two age groups of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.of Indian Central Himalaya

The populations of the general microflora (bacteria, actinomycetes and fungi) in the rhizosphere and their corresponding non-rhizosphere soil samples of Ginkgo biloba L. of two age groups (Group A, <25 years-young trees; Group B, >60 years-old trees) growing under a temperate location of Indian Himalayan Region (IHR) have been determined. Observations were also made for the diversity, distribution and colonization of arbuscular mycorrhizal (AM) fungi and occurrence of endophytes in roots of G. biloba. The population of general microflora was found to be higher in the rhizosphere of Group B trees, more clearly reflected in terms of rhizosphere: soil (R:S) ratios. Contrary to this, per cent colonization and spore densities of AM fungi were higher in the rhizosphere of Group A trees as compared to the rhizosphere of Group B. AM fungal colonization was observed mostly in form of loose coils. All the spores detected, belonged to the genus Glomus with five different types. Presence of endophytes (both bacteria and fungi) was observed in the cortical cells of G. biloba roots, more profound in case of Group B trees. Data suggest that, while the species of Glomus dominated the rhizosphere of G. biloba, an inverse correlation exist between the colonization of general microflora and the colonization of AM fungi including endophytes.     

Endophytic bacterial diversity in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR

Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with γ-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.     

Endophytic fungi from leaves of Centella asiatica: occurrence and potential interactions within leaves

Fungal endophytes were isolated from leaves of Centella asiatica (Apiaceae) collected at Mangoro (middle eastern region of Madagascar, 200 km from Antananarivo). Forty- five different taxa were recovered. The overall foliar colonization rate was 78%. The most common endophytes were the non-sporulating species 1 (isolation frequency IF 19.2%) followed by Colletotrichum sp.1 (IF 13.2%), Guignardia sp. (IF 8.5%), Glomerella sp. (IF 7.7%), an unidentified ascomycete (IF 7.2%), the non-sporulating species 2 (IF 3.7%) and Phialophora sp. (IF 3.5%). Using sequences of the ribosomal DNA internal transcribed spacer (ITS) regions, major endophytes (IF > 7%) were identified as xylariaceous taxa or as Colletotrichum higginsianum, Guignardia mangiferae and Glomerella cingulata. Results from in vitro fungal disk experiments showed a strong inhibitory activity of the xylariaceous non-sporulating species 1 against G. mangiferae and C. higginsianum and of C. higginsianum against G. mangiferae. This can be explained by antagonism between dominant taxa.     

桐花树内生和根际细菌多样性及抗血栓活性研究

红树植物内生菌在红树共生体的物质循环、能量传递和健康维护等方面起着重要作用。为探究红树植物内生菌的多样性,进一步揭示内生菌在红树共生体的功能多样性提供菌种资源,该研究选择6种分离培养基和采用传统稀释涂布法对从广西北海滩涂上采集的桐花树组织和根际土壤样品进行分离,对获得的可培养细菌进行多样性分析,并通过体外溶栓实验筛选出具有抗血栓活性的菌株。结果表明:(1)基于16S rRNA基因序列系统进化分析,从桐花树组织和根际土壤中共获得125株细菌;分布于变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes) 3个门27个科39个属74个种中,芽孢杆菌属为优势菌属,菌株数量占13.5%。(2)抗血栓活性实验表明,初筛获得18株具有抗血栓活性细菌,总阳性率为24.32%;将初筛有活性的菌株进行复筛和重复验证实验,进一步验证其活性,结果复筛出3株细菌B1850、B1989和B2632具有很强抗血栓活性。综上所述,广西北海滩涂上红树植物桐花树中存在丰富的可培养细菌资源,具有从中挖掘新的纤溶酶和开发溶血栓药物的潜力。     

药用植物青蒿不同种类的内生菌抑菌活性分析

为了研究青蒿不同种类的内生菌抑制细菌和抑制真菌的活性,该研究采用组织块法和研磨法从青蒿的根、茎、叶中分离内生细菌、放线菌和真菌,以大肠埃希菌(Escherichia coli)(CICC 23657)、枯草芽孢杆菌(Bacillus subtilis)(CICC 10275)、金黄色葡萄球菌(Staphylococcus aureus)(CICC 10384)、黑曲霉(Aspergillus niger)(CICC 2487)、酿酒酵母(Saccharomyces cerevisiae)(CICC 33032)为指示菌,采用琼脂块法和双层平板法检测内生菌的抑菌活性。结果表明:(1)从青蒿植株中共分离到76株内生菌,其中内生细菌19株、内生放线菌34株、内生真菌23株。从分离部位来看,56株来自于茎段、17株来自于根段、3株来自叶片。(2)内生细菌中抑菌活性菌株占总菌株的比例最高,为95%,内生放线菌和内生真菌中抑菌活性菌株的比例分别为41%、35%。(3)内生细菌的抗菌谱较广;虽然内生放线菌的抗菌谱较窄,但其中高抗菌株较多,尤其对酿酒酵母的抑菌效果好。综上结果显示,药用植物青蒿中存在着丰富的有抑菌活性的内生菌,且不同种类的内生菌抑菌活性不同。     

Taxonomic characterization of Shiraia-like fungi isolated from bamboos in Japan

The molecular phylogeny of nuclear LSU rDNA sequences (D1/D2 domain), ITS regions, and beta-tubulin gene (tub2) showed that the seven strains of Shiraia-like fungi obtained from fresh bamboo tissues as endophytes were closely related to Shiraia bambusicola and had three distinctive lineages (groups A-C). The closest group (group A) to S. bambusicola produced distinctive prawn-shaped conidioma-like structures that differed from conidiomata in the anamorph of S. bambusicola. Currently, none of the morphological structures and molecular database records were compatible with our Shiraia-like fungi. These results reveal that Shiraia-like fungi group A is supposed to be a new species that should be assigned into a novel genus/species related to S. bambusicola.     

Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3–65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.